This application claims the benefit of U.S. Provisional Application No. 62/563,334, filed September 26, 2017, the entire disclosure of which is incorporated by reference.

BACKGROUND OF THE INVENTION

Obtaining chromatographic retention of acidic, polar analytes such as organic acids, sugars, and phosphorylated compounds can prove difficult with current chromatographic techniques. Current methodologies often incorporate the use of ion-pair reagents, (Luo et al, Journal of Chromatography B, 1147, 2007, 153-164 and Lu et al, Analytical Chemistry, 2010, 82, 3212-3221), ion chromatography (IC) or hydrophilic interaction chromatography (HILIC) . (Bajad et al, Journal of Chromatography A, 1125, 2006, 76-88). In addition, the use of derivatization may be carried out as a means to reduce the polarity of molecules (Tan et al, Analytical Biochemistry, 465, 2014, 134-147) and to thereby make it possible to retain, separate and subsequently detect these analytes for the purposes of quantitative or qualitative analyses.

However, these current methods can suffer negative results due to sample or diluent restrictions, needing specialized equipment, or an incompatibility with mass spectrometric (MS) detection.

Thus, there remains a need for alternative materials and methods that provide enhanced retention and selectivity for acidic analytes such that it might be possible to better facilitate their analysis by LC and LC-MS.

SUMMARY OF THE INVENTION

The present invention provides the use of charged surface reversed phase

chromatographic materials along with standard reversed-phase LC and mass spectrometry compatible conditions for the retention, separation, purification, and characterization of acidic, polar molecules, including, but not limited to, organic acids, a-amino acids, phosphate sugars, nucleotides, other acidic, polar biologically relevant molecules. Improved methodologies in the analysis of these compounds is of importance to researchers, the medical community and pharmaceutical companies due to the direct involvement of these molecules in numerous disease states, such as cancer and diabetes. Further, the analysis of these molecules is of interest for the manufacturing of various products from bioreactors. (Hinder et al, Journal of Endrocrinology, 213, 2013, 1-11 and Rustin et al, Biochimica et Biophysica Acta, 1361, 1997, 185-197.)

In one aspect, the invention provides, a high purity chromatographic material (HPCM) comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety.

In certain aspects the HPCM may further comprise a chromatographic core material. In some aspects, the chromatographic core is a silica material; a hybrid inorganic/organic material; or a superficially porous material.

In another aspect the ionizable modifier contains a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, a boronic acid group, an amino group, an imido group, an amido group, a pyridyl group, an imidazolyl group, an ureido group, a thionyl-ureido group or an aminosilane group. And in one aspect, the ionizable modifier contains diethylaminopropyl group.

In another aspect, the ionizable modifier is selected from the group of zirconium, aluminum, cerium, iron, titanium, salts thereof, oxides and combinations thereof.

In another aspect, the ionizable modifier is provided by reacting the chromatographic surface with an ionizable modifying reagent selected from groups having formula (I)

the formula (II): the formula (III):

or a combination thereof wherein m is an integer from 1-8; v is 0 or 1 ; when v is 0, m' is 0; when v is 1, m' is an integer from 1-8;

Z represents a chemically reactive group, including (but not limited to)

, -OH, -OR , amine, alkylamine, dialkylamine, isocyanate, acyl chloride, triflate, isocyanate, thiocyanate, imidazole carbonate, NHS-ester, carboxylic acid, ester, epoxide, alkyne, alkene, azide, -Br, -CI, or -I;

Y is an embedded polar functionality; each occurrence of R ¹ independently represents a chemically reactive group on silicon, including (but not limited to) -H, -OH, -OR ⁶ , dialkylamine, triflate, Br, CI, I, vinyl, alkene, or -(CH2)m"Q; each occurrence of Q is -OH, -OR ⁶ , amine, alkylamine, dialkylamine, isocyanate, acyl chloride, triflate, isocyanate, thiocyanate, imidazole carbonate, NHS-ester, carboxylic acid, ester, epoxide, alkyne, alkene, azide, -Br, -CI, or -I; m" is an integer from 1 -8; p is an integer from 1-3; each occurrence of R ¹ independently represents F, C1-C18 alkyl, C2-C18 alkenyl, C2- Ci8 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or Ci- Ci8 heteroaryl, fluoroalkyl, or fluoroaryl; each occurrence of R ² , R ² , R ³ and R independently represents hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl, -Z, or a group having the formula -Si(R')bR" _a or -

a and b each represents an integer from 0 to 3 provided that a + b = 3;

R' represents a Ci - C6 straight, cyclic or branched alkyl group;

R" is a functionalizing group selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, a cation or anion exchange group, an alkyl or aryl group containing an embedded polar functionality and a chiral moiety.

R ⁴ represents hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl;

R ⁵ represents hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl; each occurrence of R ⁶ independently represents C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl; Het represents a heterocyclic or heteroaryl ring system comprising at least one nitrogen atom; and

A represents an acidic ionizable modifier moiety or a dual charge ionizable modifier moiety. In certain aspects, where the ionizable modifying reagent is selected from formula

(III), A represents a protected or unprotected alkyl, aryl, or arylalkyl groups containing phosphoric, carboxylic, sulfonic, or boronic acid.

In certain other aspects, where the ionizable modifying reagent is selected from formula (III), A represents a dual charge ionizable modifier. While not limited to theory; the dual charge ionizable modifier has two sub-groups that can display opposite charges. Under some conditions the dual charge ionizable modifier can act similarly to a zwitterions and ampholytes to display both a positive and negative charge and maintain a zero net charge. Under other conditions the dual charge ionizable may only have one group ionized and may display a net positive or negative charge.

Dual charge ionizable modifying reagents include, but are not limited to, alkyl, branched alkyl, aryl, cyclic, polyaromatic, polycyclic, hertocyclic and polyheterocyclic groups that can display a positive charge (commonly on a nitrogen or oxygen atom), and a negative charge through an acidic group that includes a carboxylic, sulfonic, phosphonic or boronic acid. Alternatively, some metal containing complexes can display both positive and negative charges.

Dual charge ionizable modifying reagents may also include, but are not limited to Zwitterion, ampholyte, amino acid, aminoalkyl sulfonic acid, aminoalkyl carboxylic acid, mono and di-methylaminoalkyl sulfonic acid, mono and di-methylaminoalkyl carboxylic acid, pyridinium alkyl sulfonic acid, and pyridinium alkyl carboxylic acid groups.

Alternatively the dual charge ionizable modifier may include 2-(N- morpholino)ethanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, 4-(2-hydroxyethyl)- 1-piperazine ethanesulfonic acid, piperazine-N,N'-bis(2-ethanesulfonic acid), N-cyclohexyl- 3-aminopropanesulfonic acid, N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid, 3-[(3- cholamidopropyl)dimethylammonio]-l -propanesulfonate, 6-Methyl-9, 10-didehydro-ergoline- 8-carboxylic acid, phenolsulfonphthalein, betaine, quinonoid, N,N-bis(2- hydroxyethyl)glycine, and N-[tris(hydroxymethyl)methyl]glycine groups. In certain aspects, where the ionizable modifying reagent is selected from formulas (I), (II) or (III),

m is 2 or 3.

In some aspects, where the ionizable modifying reagent is selected from formulas (I), (II) or (III), R ¹ represents CI, -OH, dialkylamino, methoxy or ethoxy.

In certain aspects, where the ionizable modifying reagent is selected from formulas

(I) , (II) or (III), R ¹ represents, methyl, ethyl, isobutyl, isopropyl or tert-butyl.

In other aspects where the ionizable modifying reagent is selected from formulas (I),

(II) or (III), each occurrence of R ² and R ³ represents hydrogen.

In other aspects where the ionizable modifying reagent is selected from formulas (I),

(II) or (III), each occurrence of R ² and R represents hydrogen.

In other aspects where the ionizable modifying reagent is selected from formula (I), each of R ⁴ and R ⁵ represents hydrogen.

In still other aspects where the ionizable modifying reagent is selected from formulas (II), Het is pyridyl, pyrimidinyl, pyridazinyl, pyrazinyl, piperidinyl, piperizinyl,

hexahydropyrimidinyl, pyrrolyl, pyrazolyl, imidazolyl, pyrrolidinyl, pyrazolidinyl, imidazolidinyl or triazinyl.

In other aspects where the ionizable modifying reagent is selected from formulas (I),

(II) or (III), V is 1, m' is 3, and each occurrence of R ² , R ² , R ³ and R is hydrogen. In certain aspects, where the ionizable modifying reagent is selected from formulas (I), (II) or (III), V is

1, m' is 3, and each occurrence of R ² , R ² , R ³ and R is hydrogen, Y is carbamate, carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate or triazole.

In yet other aspects, the ionizable modifying reagent is aminopropyltriethoxysilane, aminopropyltrimethoxysilane, 2-(2-(trichlorosilyl)ethyl)pyridine, 2-(2-

(trimethoxy)ethyl)pyridine, 2-(2-(triethoxy)ethyl)pyridine, 2-(4-pyridylethyl)triethoxysilane,

2-(4-pyridylethyl)trimethoxysilane, 2-(4-pyridylethyl)trichlorosilane,

chloropropyltrimethoxysilane, chloropropyltrichlorosilane, chloropropyltrichlorosilane, chloropropyltriethoxysilane, imidazolylpropyltrimethoxysilane,

imidazolylpropyltri ethoxy silane, imidazolylpropyl trichlorosilane, sulfopropyltrisilanol, carboxyethylsilanetriol, 2-(carbomethoxy)ethylmethyldichlorosilane, 2-

(carbomethoxy)ethyltrichlorosilane, 2-(carbomethoxy)ethyltrimethoxysilane, n-

(trimethoxysilylpropyl)ethylenediamine triacetic acid, (2- diethylphosphatoethyl)triethoxysilane, 3-mercaptopropyltriethoxysilane, 3- mercaptopropyltrimethoxysilane, bis[3-(triethoxysilyl)propyl]disulfide, bis[3- (triethoxysilyl)propyl]tetrasulfide, 2,2-dimethoxy- 1 -thia-2-silacy clopentane,

bis(trichlorosilylethyl)phenylsulfonyl chloride, 2-(chlorosulfonylphenyl)ethyltrichlorosilane, 2-(chlorosulfonylphenyl)ethyltrimethoxysilane, 2- (ethoxysulfonylphenyl)ethyltrimethoxysilane, 2-

(ethoxysulfonylphenyl)ethyltrimethoxysilane, 2-(ethoxysulfonylphenyl)ethyltrichlorosilane, sulphonic acid phenethyltrisilanol, (triethoxysilyl ethyl)phenyl phosphonic acid diethyl ester, (trimethoxysilyl ethyl)phenyl phosphonic acid diethyl ester, (trichlorosilyl ethyl)phenyl phosphonic acid diethyl ester, phosphonic acid phenethyltrisilanol, N-(3- trimethoxysilylpropyl)pyrrole, N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole,

bis(methyldimethoxysilylpropyl)-N-methylamine, tris(triethoxysilylpropyl)amine, bis(3- trimethoxysilylpropyl)-N-methylamine, (N,N-diethyl-3-aminopropyl)trimethoxysilane, N- (hydroxyethyl)-N-methylaminopropyltrimethoxysilane, 3-(N,N- dimethylaminopropyl)trimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, N,N'-bis(hydroxyethyl)-N,N'-bis(trimethoxysilylpropyl)ethyle nediamine, or N,N-dimethyl-3- aminopropylmethyldimethoxysilane.

In another aspect, the ionizable modifying reagent is a tris-silyl or bis-silyl compound, for instance a so-called 'bridging' silane such as an amine-containing and -bridging silanizing reagent (e.g., a molecule containing two or three silane moieties bridged by an amine moiety), for example, a bis-silylamine or a tris-silylamine. In some embodiments, the bis-silylamine or tris-silylamine may be a bis(trialkoxysilylalklyl)amine or a

tris(trialkoxysilylalklyl)amine, such as a bis(tri-C l-C4-alkoxysilyl-Cl -C4-alklyl)amine or tris(tri-Cl -C4-alkoxysilyl-Cl -C4-alklyl)amine, wherein the preceding amines can be monoamines, diamines, triamines, tetraamines, etc., including but not limited to bis(3-

trimethoxysilylpropyl)-N-methylamine,

(hydroxy ethyl)-N,N-bis(trimethoxysilylp ropyl) tris(triethoxysilylmethyl)amine, , and N,N'-bis(2-hydroxyethyl)-N,N'-

bis(trimethoxysilylpropyl)ethylenedi amine,

some aspects, these reagents are methoxy, ethoxy, chloro or dimethylamino activated silanes.

In some embodiments, the ionizable modifying reagent is a bis-silylamine or a tris- silylamine of the formula, the A(SiZiZ2Z3)n where A designates an amine (including monoamines, diamines, triamines, tetraamines, etc.), n=l or 2, and Ζι, Z2 and Z3 are independently selected from CI, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C8 alkyl, although at most two of Zi, Z2 and Z3 can be C1-C8 alkyl. More broadly, the ionizable modifying reagent may be of the formula A(Si ZiZ2Z3)q(SiZ4Z5Z6)r where q=l or 2, r=l or 2, and q+r=2 or 3, and where Z ₄ , 7S and Ζβ are independently selected from CI, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C8 alkyl, although at most two of Z ₄ , Z5 and Ζβ can be C1-C8 alkyl, or of the formula A(Si ZiZ2Z3) _s (SiZ4Z5Z6) _s (SiZ7Z8Z9) _s where s=l and where Z ₇ , Z8 and Z9 are independently selected from CI, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C8 alkyl, although at most two of Z ₇ , 7% and Z9 can be C1-C8 alkyl.

In one aspect, the ionizable modifying reagent contains a diethylaminopropyl (DEAP) group.

In another aspect, the ionizable modifying reagent contains a diethylaminopropyl (DEAP) group, and the eluting of the adsorbed acidic, polar molecule from the high purity chromatographic material is performed at 7 < pH < 10.

In yet another aspect, the ionizable modifying reagent contains a diethylaminopropyl (DEAP) group, and the eluting of the adsorbed acidic, polar molecule from the high purity chromatographic material is performed with an initial pH at 7 < pH < 10, and the pH shift during the eluting the adsorbed acidic, polar molecule from the high purity chromatographic material. In another aspect, the ionizable modifier is an amine-containing and bridging silanizing reagent and elution of the adsorbed acidic, polar molecule from the high purity chromatographic material occurs at 7 < pH < 10.

In another aspect, the ionizable modifier is an amine-containing and bridging silanizing reagent, and the acidic, polar molecule is adsorbed to the high purity

chromatographic material at a pH of 2.5 < pH < 10 and is then eluted by means of an upward shift in pH.

In other aspects, the acidic, polar molecule is eluted from the high purity

chromatographic material with weakly acidic mobile phases at 2.5 < pH < 7, including but not limited to mobile phases comprised of 0.01 to 0.5% formic acid, 1 to 50 mM ammonium formate and 1 to 50 mm ammonium acetate or combinations thereof. Elution can be initiated by either a gradient or isocratic separation. Elution may or may not entail a change in ionic strength and conductivity.

In other aspects, the ionizable modifying reagent contains a pyridylethyl group or diethylaminopropyl (DEAP) group and elution of the adsorbed acidic, polar molecule from the high purity chromatographic material is performed with weakly acidic mobile phases at 2.5 < pH < 7, including but not limited to mobile phases comprised of 0.01 to 0.5% formic acid, 1 to 50 mM ammonium formate and 1 to 50 mm ammonium acetate or combinations thereof. Elution can be initiated by either a gradient or isocratic separation. Elution may or may not entail a change in ionic strength and conductivity.

In some aspects, the ratio of the hydrophobic surface group: ionizable modifier in the HPCM of the invention is from about 2.5 : 1 to about 350: 1 ; from about 3: 1 to about 200: 1 ; from about 4: 1 to about 150: 1 ; from about 4: 1 to about 35 : 1 ; from about 5 : 1 to about 25 : 1 ; from about 5: 1 to about 22: 1 ; from about 20: 1 to about 100: 1 ; or from about 25 : 1 to about 100: 1.

In other aspects, the concentration of ionizable modifier in the HPCM of the invention is less than about 0.7 μιηοΐ/ιη ² ; less than about 0.6 μιηοΐ/ιη ² ; less than about 0.4 μιηοΐ/ιη ² ; from about 0.01 μιηο1/ιη ² ΐΌ about 0.5 μιηοΐ/ιη ² ; from about 0.01 μιτιοι/ιη ² ΐΌ about 0.4 μιηοΐ/m ² ; or from about 0.03 μιτιοι/ιη ² ΐΌ about 0.4 μιηοΐ/ιη ² .

In another aspect, the hydrophobic surface group of the HPCM of the invention is a

C4 to C30 bonded phase. In certain aspects, the hydrophobic surface group is a Cie bonded phase. In other aspects, the hydrophobic surface group is an aromatic, phenylalkyl, fluoro- aromatic, phenylhexyl, pentafluorophenylalkyl or chiral bonded phase. In still other aspects, the hydrophobic surface group is an embedded polar bonded phase.

In certain aspects, the HPCM of the invention may be in the form of a particle, a granular material, a monolith, a superficially porous material, a superficially porous particle, a superficially porous monolith, or a superficially porous layer for open tubular

chromatography.

In certain aspects, the HPCM of the invention may be in inorganic material (e.g. , silica, alumina, titania, zirconia), a hybrid organic/inorganic material, an inorganic material (e.g. , silica, alumina, titania, zirconia) with a hybrid surface layer, a hybrid material with an inorganic (e.g. , silica, alumina, titania, zirconia) surface layer, or a hybrid material with a different hybrid surface layer. In other aspects, the HPCM of the invention may have ordered pore structure, non-periodic pore structuring, non-crystalline or amorphous pore structuring or substantially disordered pore structuring.

In one aspect, the HPCM of the invention does not have chromatographically enhancing pore geometry.

In another aspect, the HPCM of the invention has chromatographically enhancing pore geometry.

In certain aspects, the HPCM of the invention has a surface area of about 25 to 1100 m ² /g; about 80 to 500 m ² /g; or about 120 to 330 m ² /g.

In other aspects, the HPCM of the invention has a pore volume of about 0.15 to 1.5 cm ² /g; or about 0.5 to 1.3 cm ² /g.

In yet other aspects, the HPCM of the invention has a micropore surface area of less than about 110 m ² /g; less than about 105 m ² /g; less than about 80 m ² /g; or less than about 50 m ² /g.

In still yet other aspects, the HPCM of the invention has an average pore diameter of about 20 to 1500 A; about 50 to ΙΟΟθΑ; about 100 to 75θΑ; or about 110 to 500 A.

In still yet other aspects, when the HPCM of the invention is in the form of a particle, the HPCM of the invention has an average particle size of about 0.3-100 μιτι; about 0.5-20 μιτι; 0.8-10 μιτι; or about 1.0-3.5 μιτι.

In another aspect, the HPCM of the invention is hydrolytically stable at a pH of about

1 to about 14; at a pH of about 10 to about 14; or at a pH of about 1 to about 5.

In still another aspect, the HPCM of the invention has a quantified surface coverage ratio, B/A, from about 2.5 to about 300 wherein A represents the ionizable modifier and B represents the hydrophobic group. In certain aspects, the quantified surface coverage ratio, B/A, is from about 3 to about 200, from about 4 to about 35 or from about 5 to about 22.

In another aspect, the HPCM of the invention may be surface modified. In certain aspects, the HPCM of the invention may be surface modified by coating with a polymer. In other aspects, the HPCM of the invention may be surface modified by coating with a polymer by a combination of organic group and silanol group modification; by a combination of organic group modification and coating with a polymer; or by a combination of silanol group modification and coating with a polymer. In other aspects, the HPCM of the invention may be material has been surface modified by a combination of organic group modification, silanol group modification and coating with a polymer. In still other aspects, the HPCM of the invention may be surface modified via formation of an organic covalent bond between the material's organic group and the modifying reagent.

In one aspect, the HPCM have a chromatographic surface containing a

diethylaminopropyl (DEAP) ionizable modifier and a Cie hydrophobic group. In another aspect, the HPCM is endcapped on a bridged ethylene hybrid particle.

In certain aspects, the HPCM of the invention may further comprising a nanoparticle dispersed within the material. In aspects further comprising a nanoparticle, the nanoparticle may be a mixture of more than one nanoparticle. In some aspects comprising a nanoparticle, the nanoparticle is present in <20% by weight of the nanocomposite or in <5% by weight of the nanocomposite. In other aspects comprising a nanoparticle, the nanoparticle is crystalline or amorphous. In certain aspects, the nanoparticle is a substance which comprises one or more moieties selected from the group consisting of silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, oxides thereof, and nitrides thereof. In certain other aspects, the nanoparticle is a substance which comprises one or more moieties selected from the group consisting of nano-diamonds, silicon carbide, titanium dioxide, cubic-boronitride. In another aspect, the nanoparticles are less than or equal to 200 nm in diameter; less than or equal to 100 nm in diameter; less than or equal to 50 nm in diameter; or less than or equal to 20 nm in diameter.

In one aspect, the invention provides a method for mixed mode, anion exchange reversed liquid chromatography and the selective retention of acidic, polar molecules from a sample. In another aspect, the invention provides a method for selectively isolating an acidic, polar molecule from a sample, the method comprising the steps of:

a) loading a sample containing an acidic, polar molecule onto a chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers such that the acidic, polar molecule is selectively adsorbed onto the high purity chromatographic material, with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety; and b) eluting the adsorbed acidic, polar molecule from the high purity chromatographic material, thereby selectively isolating the acidic, polarmolecule from the sample.

In still another aspect, the invention provides a method for separating a plurality of acidic, polarmolecules from a sample, the method comprising the steps of:

a) loading a sample containing a plurality of acidic, polar molecules onto

chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers such that the acidic, polar molecules are adsorbed onto the high purity chromatographic material, with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety; and

b) eluting the adsorbed acidic, polar molecules from the high purity chromatographic material, thereby separating the acidic, polar molecules.

In yet another aspect, the invention provides a method for purifying an acidic, polar molecule contained in a sample, the method comprising the steps of:

a) loading a sample containing an acidic, polar molecule onto chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers such that the acidic, polar molecule are adsorbed onto the high purity chromatographic material, with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety; and b) eluting the adsorbed acidic, polar molecule from the high purity chromatographic material, thereby purifying an acidic, polar molecule.

In still yet another aspect, the invention provides a method for detecting an acidic, polar molecule in a sample, the method comprising the steps of:

a) loading a sample containing an acidic, polar molecule onto chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers such that the acidic, polar molecules are adsorbed onto the high purity chromatographic material, with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety; and

b) eluting the adsorbed acidic, polar molecule from the high purity chromatographic material; and

c) detecting the acidic, polarmolecule.

In certain aspects of the chromatographic methods of the invention, the acidic, polar molecule is selected from the group consisting of organic acids, a-amino acids, phosphate sugars, nucleotides, phosphonates, glyphosate, polar pesticides and other acidic, polar biologically relevant molecules, and mixtures thereof.

In certain embodiments of the chromatographic methods of the invention, the chromatographic separations device utilized in the method is a device is selected from the group consisting of a chromatographic column, a thin layer plate, a filtration membrane, a microfluidic separation device, a sample cleanup device, a solid support, a solid phase extraction device, a microchip separation device, and a microtiter plate.

In other aspects of the chromatographic methods of the invention, a second dimension is utilized to prepare the sample or to further purify, isolate, or separate the acidic, polar molecules. In such aspects, the methods of the invention further comprise the step of preparing the sample for use in the methods by treating a mother sample to a secondary chromatographic means to obtain the sample. Alternatively, or in addition, the methods of the invention further comprise the step of treating the acidic, polar molecules eluted in the application of the methods of the invention with a secondary chromatographic means to further isolate, purify, or separate the acidic, polar molecules. In such aspects, the secondary chromatographic means may be a second chromatographic separations device comprising a chromatographic material other than a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers. In other such aspects, the secondary chromatographic means may be a second chromatographic material comprised by chromatographic separations device utilized in the methods of the invention other than a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers. In those aspects in which a secondary chromatographic separations device is utilized, such a device is selected from the group consisting of a chromatographic column, a thin layer plate, a filtration membrane, a microfluidic separation device, a sample cleanup device, a solid support, a solid phase extraction device, a microchip separation device, and a microtiter plate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 depicts the drift with pH switching (from pH 3 to pH 10) using (a) a traditional, commercial C18 bonded material and (b) the material of the instant invention.

Figure 2 depicts the peak shape of various analytes using (a) a traditional, commercial CI 8 bonded material and (b) the material of the instant invention.

Figure 3 depicts a comparison of isocratic loading behavior for amitriptyline on 4.6 x 150 mm columns containing three different HPCM CI 8 materials: (a) Product 2e which has a high level of ionizable modifier shows fronting/ Anti-Langmuirian peak shape suggesting a concave Langmuirian isotherm; (b) Product 2d which has a balanced level of ionizable modifier shows nearly symmetrical Gaussian/linear peak shape suggesting a linear

Langmuirian isotherm; and (c) Product 2b which has a very low level of ionizable modifier shows tailing/Bi-Langmuirian peak shape suggesting a convex Langmuirian isotherm.

Figure 4 depicts a comparison of isocratic loading behavior for amitriptyline on C18 columns (both 2.1 x 50 mm).

Figure 5 depicts MRM chromatograms of various TCA cycle metabolites and intermediates and the effectiveness of a mixed mode separation as performed with a DEAP HPCM column versus a Waters ACQUITY UPLC CSH C18 column of the same chromatographic particle size and column dimensions.

Figure 6 depicts MRM chromatograms of various sugar phosphates and a demonstration of the effectiveness of a mixed mode separation as performed with a DEAP HPCM column versus a Waters ACQUITY UPLC CSH C18 column of the same chromatographic particle size and column dimensions.

Figure 7 depicts MRM chromatograms of various acidic, polar, biologically-relevant small molecules and a demonstration of the effectiveness of a mixed mode separation as performed with a DEAP HPCM column versus a Waters ACQUITY UPLC CSH CI 8 column of the same chromatographic particle size and column dimensions.

Figure 8 depicts chromatograms of glyphosate and other polar pesticide using a Waters ACQUITY UPLC I-Class LC system with a DEAP HPCM column coupled with a Xevo TQ S tandem quadrupole mass spectrometer operated in ESI negative mode and in MRM acquisition mode.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel chromatographic materials, e.g. , for chromatographic separations, processes for their preparation and separations devices containing the chromatographic material. The present invention will be more fully illustrated by reference to the definitions set forth belows.

Definitions

"High Purity" or "high purity chromatographic material" includes a material which is prepared form high purity precursors. In certain aspects, high purity materials have reduced metal contamination and/or non-diminished chromatographic properties including, but not limited to, the acidity of surface silanols and the heterogeneity of the surface.

"Chromatographic surface" includes a surface which provides for chromatographic separation of a sample. In certain aspects, the chromatographic surface is porous. In some aspects, a chromatographic surface may be the surface of a particle, a superficially porous material or a monolith. In certain aspects, the chromatographic surface is composed of the surface of one or more particles, superficially porous materials or monoliths used in combination during a chromatographic separation. In certain other aspects, the

chromatographic surface is non-porous.

"Ionizable modifier" includes a functional group which bears an electron donating or electron withdrawing group. In certain aspects, the ionizable modifier contains one or more carboxylic acid groups, amino groups, imido groups, amido groups, pyridyl groups, imidazolyl groups, ureido groups, thionyl-ureido groups or aminosilane groups, or a combination thereof. In other aspects, the ionizable modifier contains a group bearing a nitrogen or phosphorous atom having a free electron lone pair. In certain aspects, the ionizable modifier is covalently attached to the material surface and has an ionizable group. In some instances it is attached to the chromatographic material by chemical modification of a surface hybrid group.

"Hydrophobic surface group" includes a surface group on the chromatographic surface which exhibits hydrophobicity. In certain aspects, a hydrophobic group can be a carbon bonded phase such as a C4 to CI 8 bonded phase. In other aspects, a hydrophobic surface group can contain an embedded polar group such that the external portion of the hydrophobic surface maintains hydrophobicity. In some instances it is a attached to the chromatographic material by chemical modification of a surface hybrid group. In other instances the hydrophobic group can be C4-C30, embedded polar, chiral, phenylalkyl, or pentafluorophenyl bonding and coatings.

"Chromatographic core" includes a chromatographic materials, including but not limited to an organic material such as silica or a hybrid material, as defined herein, in the form of a particle, a monolith or another suitable structure which forms an intemal portion of the materials of the invention. In certain aspects, the surface of the chromatographic core represents the chromatographic surface, as defined herein, or represents a material encased by a chromatographic surface, as defined herein. The chromatographic surface material may be disposed on or bonded to or annealed to the chromatographic core in such a way that a discrete or distinct transition is discemable or may be bound to the chromatographic core in such a way as to blend with the surface of the chromatographic core resulting in a gradation of materials and no discrete internal core surface. In certain embodiments, the

chromatographic surface material may be the same or different from the material of the chromatographic core and may exhibit different physical or physiochemical properties from the chromatographic core, including, but not limited to, pore volume, surface area, average pore diameter, carbon content or hydrolytic pH stability

"Hybrid", including "hybrid inorganic/organic material," includes inorganic-based structures wherein an organic functionality is integral to both the internal or "skeletal" inorganic structure as well as the hybrid material surface. The inorganic portion of the hybrid material may be, e.g., alumina, silica, titanium, cerium, or zirconium or oxides thereof, or ceramic material. "Hybrid" includes inorganic-based structures wherein an organic functionality is integral to both the internal or "skeletal" inorganic structure as well as the hybrid material surface. As noted above, exemplary hybrid materials are shown in U.S. Patent Nos. 4,017,528, 6,528,167, 6,686,035 and 7,175,913.

The term "alicyclic group" includes closed ring structures of three or more carbon atoms. Alicyclic groups include cycloparaffins or naphthenes which are saturated cyclic hydrocarbons, cycloolefins, which are unsaturated with two or more double bonds, and cycloacetylenes which have a triple bond. They do not include aromatic groups. Examples of cycloparaffins include cyclopropane, cyclohexane and cyclopentane. Examples of cycloolefins include cyclopentadiene and cyclooctatetraene. Alicyclic groups also include fused ring structures and substituted alicyclic groups such as alkyl substituted alicyclic groups. In the instance of the alicyclics such substituents can further comprise a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower

alkylcarboxyl, a nitro, a hydroxyl, -CF3, -CN, or the like.

The term "aliphatic group" includes organic compounds characterized by straight or branched chains, typically having between 1 and 22 carbon atoms. Aliphatic groups include alkyl groups, alkenyl groups and alkynyl groups. In complex structures, the chains can be branched or cross-linked. Alkyl groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups and branched-chain alkyl groups. Such hydrocarbon moieties may be substituted on one or more carbons with, for example, a halogen, a hydroxyl, a thiol, an amino, an alkoxy, an alkylcarboxy, an alkylthio, or a nitro group. Unless the number of carbons is otherwise specified, "lower aliphatic" as used herein means an aliphatic group, as defined above (e.g., lower alkyl, lower alkenyl, lower alkynyl), but having from one to six carbon atoms. Representative of such lower aliphatic groups, e.g., lower alkyl groups, are methyl, ethyl, n-propyl, isopropyl, 2-chloropropyl, n-butyl, sec-butyl, 2-aminobutyl, isobutyl, tert-butyl, 3-thiopentyl and the like. As used herein, the term "nitro" means -N02; the term "halogen" designates -F, -CI, -Br or -I; the term "thiol" means SH; and the term "hydroxyl" means -OH. Thus, the term "alkylamino" as used herein means an alkyl group, as defined above, having an amino group attached thereto. Suitable alkylamino groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term "alkylthio" refers to an alkyl group, as defined above, having a sulfhydryl group attached thereto. Suitable alkylthio groups include groups having 1 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms. The term "alkylcarboxyl" as used herein means an alkyl group, as defined above, having a carboxyl group attached thereto. The term "alkoxy" as used herein means an alkyl group, as defined above, having an oxygen atom attached thereto. Representative alkoxy groups include groups having 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms, e.g., methoxy, ethoxy, propoxy, tert-butoxy and the like. The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous to alkyls, but which contain at least one double or triple bond respectively. Suitable alkenyl and alkynyl groups include groups having 2 to about 12 carbon atoms, preferably from 1 to about 6 carbon atoms.

The term "alkyl" includes saturated aliphatic groups, including straight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups and cycloalkyl substituted alkyl groups. In certain embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone, e.g., C1-C30 for straight chain or C3-C30 for branched chain. In certain embodiments, a straight chain or branched chain alkyl has 20 or fewer carbon atoms in its backbone , e.g., C1-C20 for straight chain or C3-C20 for branched chain, and more preferably 18 or fewer. Likewise, preferred cycloalkyls have from 4-10 carbon atoms in their ring structure and more preferably have 4-7 carbon atoms in the ring structure. The term "lower alkyl" refers to alkyl groups having from 1 to 6 carbons in the chain and to cycloalkyls having from 3 to 6 carbons in the ring structure.

Moreover, the term "alkyl" (including "lower alkyl") as used throughout the specification and Claims includes both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxy carbonyloxy,

aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxy carbonyl, aminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino

(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfate, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl, aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. Cycloalkyls can be further substituted, e.g., with the substituents described above. An "aralkyl" moiety is an alkyl substituted with an aryl, e.g., having 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, e.g., phenylmethyl (benzyl). The term "amino," as used herein, refers to an unsubstituted or substituted moiety of the formula -NRaRb, in which Ra and Rb are each independently hydrogen, alkyl, aryl, or heterocyclyl, or Ra and Rb, taken together with the nitrogen atom to which they are attached, form a cyclic moiety having from 3 to 8 atoms in the ring. Thus, the term "amino" includes cyclic amino moieties such as piperidinyl or pyrrolidinyl groups, unless otherwise stated. An "amino-substituted amino group" refers to an amino group in which at least one of Ra and Rb, is further substituted with an amino group.

The term "aromatic group" includes unsaturated cyclic hydrocarbons containing one or more rings. Aromatic groups include 5- and 6-membered single-ring groups which may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like. The aromatic ring may be substituted at one or more ring positions with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, -CF3, -CN, or the like.

The term "aryl" includes 5- and 6-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, unsubstituted or substituted benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine and the like. Aryl groups also include poly cyclic fused aromatic groups such as naphthyl, quinolyl, indolyl and the like. The aromatic ring can be substituted at one or more ring positions with such substituents, e.g., as described above for alkyl groups. Suitable aryl groups include unsubstituted and substituted phenyl groups. The term

"aryloxy" as used herein means an aryl group, as defined above, having an oxygen atom attached thereto. The term "aralkoxy" as used herein means an aralkyl group, as defined above, having an oxygen atom attached thereto. Suitable aralkoxy groups have 1 to 3 separate or fused rings and from 6 to about 18 carbon ring atoms, e.g., O-benzyl.

The term "ceramic precursor" is intended include any compound that results in the formation of a ceramic material.

The term "chiral moiety" is intended to include any functionality that allows for chiral or stereoselective syntheses. Chiral moieties include, but are not limited to, substituent groups having at least one chiral center, natural and unnatural amino-acids, peptides and proteins, derivatized cellulose, macrocyclic antibiotics, cyclodextrins, crown ethers, and metal complexes. The term "embedded polar functionality" is a functionality that provides an integral polar moiety such that the interaction with basic samples due to shielding of the unreacted silanol groups on the silica surface is reduced. Embedded polar functionalities include, but are not limited to carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, triazole functionalities or carbamate functionalities such as disclosed in U. S. Patent No. 5,374,755, and chiral moieties.

The language "chromatographically-enhancing pore geometry" includes the geometry of the pore configuration of the presently-disclosed materials, which has been found to enhance the chromatographic separation ability of the material, e.g., as distinguished from other chromatographic media in the art. For example, a geometry can be formed, selected or constructed, and various properties and/or factors can be used to determine whether the chromatographic separations ability of the material has been "enhanced", e.g., as compared to a geometry known or conventionally used in the art. Examples of these factors include high separation efficiency, longer column life and high mass transfer properties (as evidenced by, e.g., reduced band spreading and good peak shape.) These properties can be measured or observed using art-recognized techniques. For example, the chromatographically-enhancing pore geometry of the present porous inorganic/organic hybrid materials is distinguished from the prior art materials by the absence of "ink bottle" or "shell shaped" pore geometry or morphology, both of which are undesirable because they, e.g., reduce mass transfer rates, leading to lower efficiencies.

Chromatographically-enhancing pore geometry is found in hybrid materials containing only a small population of micropores. A small population of micropores is achieved in hybrid materials when all pores of a diameter of about < 34A contribute less than about 110 m^/g to the specific surface area of the material. Hybrid materials with such a low micropore surface area (MSA) give chromatographic enhancements including high separation efficiency and good mass transfer properties (as evidenced by, e.g., reduced band spreading and good peak shape). Micropore surface area (MSA) is defined as the surface area in pores with diameters less than or equal to 34A, determined by multipoint nitrogen sorption analysis from the adsorption leg of the isotherm using the BJH method. As used herein, the acronyms "MSA" and "MP A" are used interchangeably to denote "micropore surface area".

The term "functionalizing group" includes organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase. The term "heterocyclic group" includes closed ring structures in which one or more of the atoms in the ring is an element other than carbon, for example, nitrogen, sulfur, or oxygen. Heterocyclic groups can be saturated or unsaturated and heterocyclic groups such as pyrrole and furan can have aromatic character. They include fused ring structures such as quinoline and isoquinoline. Other examples of heterocyclic groups include pyridine and purine. Heterocyclic groups can also be substituted at one or more constituent atoms with, for example, a halogen, a lower alkyl, a lower alkenyl, a lower alkoxy, a lower alkylthio, a lower alkylamino, a lower alkylcarboxyl, a nitro, a hydroxyl, -CF3, -CN, or the like. Suitable heteroaromatic and heteroalicyclic groups generally will have 1 to 3 separate or fused rings with 3 to about 8 members per ring and one or more N, O or S atoms, e.g. coumarinyl, quinolinyl, pyridyl, pyrazinyl, pyrimidyl, furyl, pyrrolyl, thienyl, thiazolyl, oxazolyl, imidazolyl, indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino and pyrrolidinyl.

The term "metal oxide precursor" is intended include any compound that contains a metal and results in the formation of a metal oxide, e.g. , alumina, silica, titanium oxide, zirconium oxide.

The term "monolith" is intended to include a collection of individual particles packed into a bed formation, in which the shape and morphology of the individual particles are maintained. The particles are advantageously packed using a material that binds the particles together. Any number of binding materials that are well known in the art can be used such as, for example, linear or cross-linked polymers of divinylbenzene, methacrylate, urethanes, alkenes, alkynes, amines, amides, isocyanates, or epoxy groups, as well as condensation reactions of organoalkoxysilanes, tetraalkoxysilanes, polyorganoalkoxysiloxanes, polyethoxysiloxanes, and ceramic precursors. In certain embodiments, the term "monolith" also includes hybrid monoliths made by other methods, such as hybrid monoliths detailed in US Patent No. 7,250,214; hybrid monoliths prepared from the condensation of one or more monomers that contain 0-99 mole percent silica (e.g., SiC ); hybrid monoliths prepared from coalesced porous inorganic/organic particles; hybrid monoliths that have a

chromatographically-enhancing pore geometry; hybrid monoliths that do not have a chromatographically-enhancing pore geometry; hybrid monoliths that have ordered pore structure; hybrid monoliths that have non-periodic pore structure; hybrid monoliths that have non-crystalline or amorphous molecular ordering; hybrid monoliths that have crystalline domains or regions; hybrid monoliths with a variety of different macropore and mesopore properties; and hybrid monoliths in a variety of different aspect ratios. In certain embodiments, the term "monolith" also includes inorganic monoliths, such as those described in G. Guiochon /J. Chromatogr. A 1168 (2007) 101-168.

The term "nanoparticle" is a microscopic particle/grain or microscopic member of a powder/nanopowder with at least one dimension less than about 100 nm, e.g., a diameter or particle thickness of less than about 100 nm (0.1 mm), which may be crystalline or noncrystalline. Nanoparticles have properties different from, and often superior to those of conventional bulk materials including, for example, greater strength, hardness, ductility, sinterability, and greater reactivity among others. Considerable scientific study continues to be devoted to determining the properties of nanomaterials, small amounts of which have been synthesized (mainly as nano-size powders) by a number of processes including colloidal precipitation, mechanical grinding, and gas-phase nucleation and growth. Extensive reviews have documented recent developments in nano-phase materials, and are incorporated herein by reference thereto: Gleiter, H. (1989) "Nano-crystalline materials," Prog. Mater. Sci.

33:223-315 and Siegel, R. W. (1993) "Synthesis and properties of nano-phase materials," Mater. Sci. Eng. A168: 189-197. In certain embodiments, the nanoparticles comprise oxides or nitrides of the following: silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, and mixtures thereof. In certain embodiments, the nanoparticles of the present invention are selected from diamonds, zirconium oxide (amorphous, monoclinic, tetragonal and cubic forms), titanium oxide (amorphous, anatase, brookite and rutile forms), aluminum (amorphous, alpha, and gamma forms), and boronitride (cubic form). In particular embodiments, the nanoparticles of the present invention are selected from nano-diamonds, silicon carbide, titanium dioxide (anatase form), cubic-boronitride, and any combination thereof. Moreover, in particular

embodiments, the nanoparticles may be crystalline or amorphous. In particular

embodiments, the nanoparticles are less than or equal to 100 mm in diameter, e.g. , less than or equal to 50 mm in diameter, e.g. , less than or equal to 20 mm in diameter.

Moreover, it should be understood that the nanoparticles that are characterized as dispersed within the composites of the invention are intended to describe exogenously added nanoparticles. This is in contrast to nanoparticles, or formations containing significant similarity with putative nanoparticles, that are capable of formation in situ, wherein, for example, macromolecular structures, such as particles, may comprise an aggregation of these endogenously created.

The term "substantially disordered" refers to a lack of pore ordering based on x-ray powder diffraction analysis. Specifically, "substantially disordered" is defined by the lack of a peak at a diffraction angle that corresponds to a d value (or d-spacing) of at least 1 nm in an x-ray diffraction pattern.

"Surface modifiers" include (typically) organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase. The porous inorganic/organic hybrid materials possess both organic groups and silanol groups which may additionally be substituted or derivatized with a surface modifier.

The language "surface modified" is used herein to describe the composite material of the present invention that possess both organic groups and silanol groups which may additionally be substituted or derivatized with a surface modifier. "Surface modifiers" include (typically) organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase. Surface modifiers such as disclosed herein are attached to the base material, e.g., via derivatization or coating and later crosslinking, imparting the chemical character of the surface modifier to the base material. In one embodiment, the organic groups of a hybrid material, react to form an organic covalent bond with a surface modifier. The modifiers can form an organic covalent bond to the material's organic group via a number of mechanisms well known in organic and polymer chemistry including but not limited to nucleophilic, electrophilic, cycloaddition, free-radical, carbene, nitrene, and carbocation reactions. Organic covalent bonds are defined to involve the formation of a covalent bond between the common elements of organic chemistry including but not limited to hydrogen, boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur, and the halogens. In addition, carbon-silicon and carbon-oxygen-silicon bonds are defined as organic covalent bonds, whereas silicon-oxygen-silicon bonds that are not defined as organic covalent bonds. A variety of synthetic transformations are well known in the literature, see, e.g., March, J. Advanced Organic Chemistry, 3rd Edition, Wiley, New York, 1985.

The term "acidic, polar molecule" includes organic acids, a-amino acids, phosphate sugars, nucleotides, phosphonates, glyphosate, polar pesticides and other acidic, polar biologically relevant molecules, or mixtures thereof. Exemplary acidic, polar molecules in accordance with the invention include succinic acid, malic acid, cis aconitate acid, nicotinic acid, glutamine, glucose 6 phosphate, fructose 6 phosphate, adenosine mono-phosphate, nicotinic acid mono nucleotide, adenosine diphosphate, glufosinate, glyphosate,

aminomethylphosphonic acid, etidronic acid and mixtures thereof. The term 'acidic, polar molecule' is also inclusive of compounds containing multiple chemical moieties where at least one of them is an acidic, polar group. Lecithin, a phospholipid, is one such exemplary molecule.

The term "mother sample" includes any sample including one or more

macromolecules, including, but not limited to, a sample derived from a biological fluid selected from the group consisting of blood, urine, spinal fluid, synovial fluid, sputum, semen, saliva, tears, gastric juices and extracts and/or dilutions/solutions thereof, which is subjected to chromatographic or other separation means prior to obtain a sample for isolation, separation, purification, or detection by the materials and methods of the invention.

The term "Chromatographic separations device" includes any device capable of performing a chromatographic separation, including, but not limited to, a chromatographic column, a thin layer plate, a filtration membrane, a microfluidic separation device, a sample cleanup device, a solid support, a solid phase extraction device, a microchip separation device, and a microtiter plate.

The term "secondary chromatographic separations" includes chromatographic separations devices and chromatographic materials comprised by chromatographic separation devices. In certain embodiments, a secondary chromatographic separations means is a separate or additional chromatographic separation device than the chromatographic separations device utilized in the methods of the invention. In other embodiments, the secondary chromatographic separations means is a separate or additional chromatographic material housed by the same chromatographic separations device utilized in the methods of the invention.

The term "mixed mode" includes any chromatographic separation in which retention of molecules is based on more than one type of interaction. An exemplary embodiment of mixed mode chromatography is anion exchange reversed phase liquid chromatography, wherein acidic molecules are retained onto a chromatographic material based on interactions with a basic ionizable modifier as well as a hydrophobic surface group.

Chromatographic Surface materials The invention provides, a high purity chromatographic material (HPCM) comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety.

In certain aspects the HPCM may further comprise a chromatographic core material. In some aspects, the chromatographic core is a silica material; a hybrid inorganic/organic material; a superficially porous material; or a superficially porous particle. The

chromatographic core material may be in the form of discreet particles or may be a monolith. The chromatographic core material may be any porous material and may be commercially available or may be produced by known methods, such as those methods described in, for example, in U. S. Patent Nos. 4,017,528, 6,528, 167, 6,686,035 and 7, 175,913. In some embodiments, the chromatographic core material may be a non-porous core.

The composition of the chromatographic surface material and the chromatographic core material (if present) may be varied by one of ordinary skill in the art to provide enhanced chromatographic selectivity, enhanced column chemical stability, enhanced column efficiency, and/or enhanced mechanical strength. Similarly, the composition of the surrounding material provides a change in hydrophilic/lipophilic balance (HLB), surface charge (e.g. , isoelectric point or silanol pKa), and/or surface functionality for enhanced chromatographic separation. Furthermore, in some embodiments, the composition of the chromatographic material may also provide a surface functionality for available for further surface modification.

The ionizable modifiers and the hydrophobic surface groups of of the HPCMs of the invention can be prepared using known methods. Some of the ionizable modifier reagents are commercially available. For example silanes having amino alkyl trialkoxysilanes, methyl amino alkyl trialkoxysilanes, and pyridyl alkyl trialkoxysilanes are commercially available. Other silanes such as chloropropyl alkyl trichlorosilane and chloropropyl alkyl

trialkoxysilane are also commercially available. These can be bonded and reacted with imidazole to create imidazolyl alkyl silyl surface species, or bonded and reacted with pyridine to create pyridyl alkyl silyl surface species. Other acidic modifiers are also commercially available, including, but not limited to, sulfopropyltrisilanol,

carboxyethylsilanetriol, 2-(carbomethoxy)ethylmethyldichlorosilane, 2- (carbomethoxy)ethyltrichlorosilane, 2-(carbomethoxy)ethyltrimethoxysilane, n- (trimethoxysilylpropyl)ethylenediamine, triacetic acid, (2- diethylphosphatoethyl)triethoxysilane, 2-(chlorosulfonylphenyl)ethyltrichlorosilane, and 2- (chlorosulfonylphenyl)ethyltrimethoxysilane.

It is known to one skilled in the art to synthesize these types of silanes using common synthetic protocols, including Grinard reactions and hydrosilylations. Products can be purified by chromatography, recrystallization or distillation

Other additives such as isocyanates are also commercially available or can be synthesized by one skilled in the art. A common isocyanate forming protocol is the reaction of a primary amine with phosgene or a reagent known as Triphosgene.

In some embodiments the ionizable modifier contains a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, a boronic acid group, an amino group, an imido group, an amido group, a pyridyl group, an imidazolyl group, an ureido group, a thionyl- ureido group or an aminosilane group.

In other aspects the ionizable modifier reagent may be selected from groups formula

(I)

the formula (II):

the formula (III): wherein m is an integer from 1-8; v is 0 or 1 ; when v is 0, m' is 0; when v is 1, m' is an integer from 1-8;

Z represents a chemically reactive group, including (but not limited to)

(R , -OH, -OR , amine, alkylamine, dialkylamine, isocyanate, acyl chloride, triflate, isocyanate, thiocyanate, imidazole carbonate, NHS-ester, carboxylic acid, ester, epoxide, alkyne, alkene, azide, -Br, -CI, or -I;

Y is an embedded polar functionality; each occurrence of R ¹ independently represents a chemically reactive group on silicon, including (but not limited to) -H, -OH, -OR ⁶ , dialkylamine, triflate, Br, CI, I, vinyl, alkene, or -(CH2)m"Q; each occurrence of Q is -OH, -OR ⁶ , amine, alkylamine, dialkylamine, isocyanate, acyl chloride, triflate, isocyanate, thiocyanate, imidazole carbonate, NHS-ester, carboxylic acid, ester, epoxide, alkyne, alkene, azide, -Br, -CI, or -I; m" is an integer from 1-8 p is an integer from 1-3; each occurrence of R ¹ independently represents F, Ci-Cie alkyl, C2-C18 alkenyl, C2- Ci8 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or Ci- Ci8 heteroaryl, fluoroalkyl, or fluoroaryl; each occurrence of R ² , R ² , R ³ and R independently represents hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C2-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C4-C18 heteroaryl, -Z, or a group having the formula -Si(R')bR" _a or -

a and b each represents an integer from 0 to 3 provided that a + b = 3;

R' represents a Ci - C6 straight, cyclic or branched alkyl group;

R" is a functionalizing group selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, a cation or anion exchange group, an alkyl or aryl group containing an embedded polar functionality and a chiral moiety.

R ⁴ represents hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl;

R ⁵ represents hydrogen, C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl; each occurrence of R ⁶ independently represents C1-C18 alkyl, C2-C18 alkenyl, C2-C18 alkynyl, C3-C18 cycloalkyl, C1-C18 heterocycloalkyl, C5-C18 aryl, C5-C18 aryloxy, or C1-C18 heteroaryl;

Het represents a heterocyclic or heteroaryl ring system comprising at least one nitrogen atom; and

A represents an acidic ionizable modifier moiety or a dual charge ionizable modifier moiety.

In yet other embodiments, the ionizable modifier is an amine-containing bis- or tris- silyl compound, a so-called bridging silane.

In yet other embodiments, the inoizable modifier is aminopropyltriethoxysilane, aminopropyltrimethoxysilane, 2-(2-(trichlorosilyl)ethyl)pyridine, 2-(2- (trimethoxy)ethyl)pyridine, 2-(2-(triethoxy)ethyl)pyridine, 2-(4-pyridylethyl)triethoxysilane, 2-(4-pyridylethyl)trimethoxysilane, 2-(4-pyridylethyl)trichlorosilane,

chloropropyltrimethoxysilane, chloropropyltrichlorosilane, chloropropyltrichlorosilane, chloropropyltriethoxysilane, imidazolylpropyltrimethoxysilane,

imidazolylpropyltriethoxysilane, imidazolylpropyl trichlorosilane, sulfopropyltrisilanol, carboxyethylsilanetriol, 2-(carbomethoxy)ethylmethyldichlorosilane, 2- (carbomethoxy)ethyltrichlorosilane, 2-(carbomethoxy)ethyltrimethoxysilane, n- (trimethoxysilylpropyl)ethylenediamine triacetic acid, (2- diethylphosphatoethyl)triethoxysilane, 3-mercaptopropyltriethoxysilane, 3- mercaptopropyltrimethoxysilane, bis[3-(triethoxysilyl)propyl]disulfide, bis[3- (triethoxysilyl)propyl]tetrasulfide, 2,2-dimethoxy- 1 -thia-2-silacy clopentane,

bis(trichlorosilylethyl)phenylsulfonyl chloride, 2-(chlorosulfonylphenyl)ethyltrichlorosilane, 2-(chlorosulfonylphenyl)ethyltrimethoxysilane, 2- (ethoxysulfonylphenyl)ethyltrimethoxysilane, 2-

(ethoxysulfonylphenyl)ethyltrimethoxysilane, 2-(ethoxysulfonylphenyl)ethyltrichlorosilane, sulphonic acid phenethyltrisilanol, (triethoxysilyl ethyl)phenyl phosphonic acid diethyl ester, (trimethoxysilyl ethyl)phenyl phosphonic acid diethyl ester, (trichlorosilyl ethyl)phenyl phosphonic acid diethyl ester, phosphonic acid phenethyltrisilanol, N-(3- trimethoxysilylpropyl)pyrrole, N-(3-triethoxysilylpropyl)-4,5-dihydroimidazole,

bis(methyldimethoxysilylpropyl)-N-methylamine, tris(triethoxysilylpropyl)amine, bis(3- trimethoxysilylpropyl)-N-methylamine, (N,N-diethyl-3-aminopropyl)trimethoxysilane, N- (hydroxyethyl)-N-methylaminopropyltrimethoxysilane, 3-(N,N- dimethylaminopropyl)trimethoxysilane, bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, N,N'-bis(hydroxyethyl)-N,N'-bis(trimethoxysilylpropyl)ethyle nediamine, or N,N-dimethyl-3- aminopropylmethyldimethoxysilane.

In certain embodiments, when the ionizable modifier is of the formula (III), the acidic ionizable modifiers is a protected or deprotected forms of trisilanol, trialkoxysilane or trichlorosilane; or a salt of sulfonic acid alkyl silanes, sulfonic acid phenylalkyl silanes, sulfonic acid benzylalkyl silanes, sulfonic acid phenyl silanes, sulfonic acid benzyl silanes, carboxylic acid alkyl silanes, carboxylic acid phenylalkyl silanes, carboxylic acid benzylalkyl silanes, carboxylic acid phenyl silanes, carboxylic acid benzyl silanes, phosphoric acid alkyl silanes, phosphonic acid phenylalkyl silanes, phosphonic acid benzylalkyl silanes, phosphonic acid phenyl silanes, phosphonic acid benzyl silanes, boronic acid alkyl silanes, boronic acid phenylalkyl silanes, boronic acid benzylalkyl silanes, boronic acid phenyl silanes, boronic acid benzyl silanes.

In certain embodiments, when the ionizable modifier is of the formula (III), the acidic ionizable modifiers is a protected or deprotected version or a salt of sulfonic acid alkyl isocyanates, sulfonic acid phenylalkyl isocyanates, sulfonic acid benzylalkyl isocyanates, sulfonic acid phenyl isocyanates, sulfonic acid benzyl isocyanates carboxylic acid alkyl isocyanates, carboxylic acid phenylalkyl isocyanates, carboxylic acid benzylalkyl isocyanates, carboxylic acid phenyl isocyanates, carboxylic acid benzyl isocyanates, phosphoric acid alkyl isocyanates, phosphonic acid phenylalkyl isocyanates, phosphonic acid benzylalkyl isocyanates, phosphonic acid phenyl isocyanates, phosphonic acid benzyl isocyanates, boronic acid alkyl isocyanates, boronic acid phenylalkyl isocyanates, boronic acid benzylalkyl isocyanates, boronic acid phenyl isocyanates, or boronic acid benzyl isocyanates.

In certain embodiments, when the inoizable modifier reagent is selected from formula (III), A represents a dual charge ionizable modifier moiety. While not limited to theory; the dual charge ionizable modifier moiety has two sub-groups that can display opposite charges. Under some conditions the dual charge ionizable modifier moiety can act similarly to a zwitterions and ampholytes to display both a positive and negative charge and maintain a zero net charge. Under other conditions the dual charge ionizable modifier moiety may only have one group ionized and may display a net positive or negative charge. Dual charge ionizable modifier moieties include, but are not limited to, alkyl, branched alkyl, aryl, cyclic, polyaromatic, poly cyclic, hertocyclic and polyheterocyclic groups that can display a positive charge (commonly on a nitrogen or oxygen atom), and a negative charge through an acidic group that includes a carboxylic, sulfonic, phosphonic or boronic acid. Alternatively, some metal containing complexes can display both positive and negative charges. Dual charge ionizable modifier moieties may also include, but are not limited to zwitterions, ampholyte, amino acid, aminoalkyl sulfonic acid, aminoalkyl carboxylic acid, mono and di- methylaminoalkyl sulfonic acid, mono and di-methylaminoalkyl carboxylic acid, pyridinium alkyl sulfonic acid, and pyridinium alkyl carboxylic acid groups. Alternatively the dual charge ionizable modifier moiety may be 2-(N-morpholino)ethanesulfonic acid, 3-(N- morpholino)propanesulfonic acid, 4-(2-hydroxyethyl)-l -piperazine ethanesulfonic acid), piperazine-N,N'-bis(2-ethanesulfonic acid), N-cyclohexyl-3-aminopropanesulfonic acid, N- cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid, 3-[(3- cholaniidopropyl)dimethylanimonio]-l-propanesulfonate, 6-Methyl-9, 1 O-didehydro-ergoline- 8-carboxylic acid, phenolsulfonphthalein, betaines, quinonoids, N,N-bis(2- hydroxyethyl)glycine, and N-[tris(hydroxymethyl)methyl]glycine groups.

In certain embodiments, the ionizable modifying reagent is a tris-silyl or bis-silyl compound, for instance a so-called 'bridging' silane such as an amine-containing and - bridging silanizing reagent (e.g., a molecule containing two or three silane moieties bridged by an amine moiety), for example, a bis-silylamine or a tris-silylamine. In some

embodiments, the bis-silylamine or tris-silylamine may be a bis(trialkoxysilylalklyl)amine or a tris(trialkoxysilylalklyl)amine, such as a bis(tri-Cl-C4-alkoxysilyl-Cl-C4-alklyl)amine or tris(tri-Cl-C4-alkoxysilyl-Cl-C4-alklyl)amine, wherein the preceding amines can be monoamines, diamines, triamines, tetraamines etc., including but not limited to bis(3-

trimethoxysilylpropyl)-N-methyl , N-

(hydroxyethyl)-N,N-bis(trimethox silylp ropyl)

tris(triethoxysilylmethyl)amine, '-bis(2-hydroxyethyl)-N,N'-

bis(trimethoxysilylpropyl)ethylenediamine, . In some aspects, these reagents are methoxy, ethoxy, chloro or dimethylamino activated silanes.

In certain embodiments, the ionizable modifying reagent is a bis-silylamine or a tris- silylamine of the formula, the A(SiZiZ2Z3)n where A designates an amine (including monoamines, diamines, triamines, tetraamines, etc.), n=l or 2, and Zi, Z2 and Z3 are independently selected from CI, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C8 alkyl, although at most two of Zi, Z2 and Z3 can be C1-C8 alkyl. More broadly, the ionizable modifying reagent may be of the formula A(Si ZiZ2Z3)q(SiZ4Z5Z6)r where q=l or 2, r=l or 2, and q+r=2 or 3, and where 7A, Z5 and Ζβ are independently selected from CI, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C8 alkyl, although at most two of Z ₄ , Z5 and Ζβ can be C1-C8 alkyl, or of the formula A(Si ZiZ2Z3) _s (SiZ4Z5Z6) _s (SiZ7Z8Z9) _s where s=l and where Z7, Z8 and Z9 are independently selected from CI, Br, I, C1-C4 alkoxy, C1-C4 alkylamino, and C1-C8 alkyl, although at most two of Z7, 7% and Z9 can be C1-C8 alkyl.

In another aspect, the ionizable modifying reagent contains a diethylaminopropyl (DEAP) group.

In some embodiments, the ratio of the hydrophobic surface group: ionizable modifier in the HPCM of the invention is from about 4: 1 to about 150: 1; from about 20: 1 to about 100: 1 ; or from about 25: 1 to about 100: 1.

In other embodiments, the concentration of ionizable modifier in the HPCM of the invention is less than about 0.7 μιηοΐ/ιη ² ; less than about 0.6 μιηοΐ/ιη ² ; less than about 0.4 μιηοΐ/m ² ; from about 0.01 μιτιοι/ιη ² ΐΌ about 0.5 μιηοΐ/ιη ² ; from about 0.1 μιτιοι/ιη ² ΐΌ about 0.4 μιηοΐ/ιη ² ; or from about 0.2 μιτιοι/ιη ² ΐΌ about 0.4 μιηοΐ/ιη ² .

In still another aspect, the HPCM of the invention has a quantified surface coverage ratio, B/A, from about 2.5 to about 300 wherein A represents the ionizable modifier and B represents the hydrophobic group. In certain aspects, the quantified surface coverage ratio, B/A, is from about 3 to about 200, from about 4 to about 35 or from about 5 to about 22.

In another aspect, the hydrophobic surface group of the HPCM of the invention is a C4 to CI 8 bonded phase. In certain aspects, the hydrophobic surface group is a CI 8 bonded phase. In still other aspects, the hydrophobic surface group is an embedded polar bonded phase. In other aspects, the hydrophobic surface group is an aromatic, phenylalkyl, fluoro- aromatic, phenylhexyl, or pentafluorophenylalkyl bonded phase. In another aspect, the hydrophobic surface group is a C4-C30, embedded polar, chiral, phenylalkyl, or

pentafluorophenyl bonding or coating.

In certain embodiments, the HPCM of the invention may be in the form of a particle, a monolith or a superficially porous material. In certain other aspects, the HPCM of the invention is a non-porous material.

In certain aspects, the HPCM of the invention may be an inorganic material (e.g., silica,), a hybrid organic/inorganic material, an inorganic material (e.g., silica) with a hybrid surface layer, a hybrid particle with a inorganic (e.g., silica) surface layer, or a hybrid particle with a different hybrid surface layer.

In yet another aspects, a HPCM with a chromatographic surface produced by a diethylaminopropyl (DEAP) ionizable modifier, a C18 hydrophobic group and endcapping on a bridged ethylene hybrid particle has proven to be an exemplary embodiment for the separation of the acidic, polar molecules noted above. This diethylaminopropyl charged surface hybrid (DEAP HPCM) stationary phase is highly effective in providing two mechanisms to aid the retention of polar acids, namely anionic exchange and hydrophobic adsorption/partitioning. It is discovered that as a result of being modified with a relatively high pKa (-10) ionizable modifier, the DEAP HPCM stationary phase yields uniquely pronounced anionic retention.

In yet another aspects, a HPCM with a chromatographic surface produced by a diethylaminopropyl (DEAP) ionizable modifier, a CI 8 hydrophobic group and endcapping on a bridged ethylene hybrid particle has proven to be an exemplary embodiment for the separation of the acidic, polar molecules below pH 10. Below pH 10, DEAP will be charged, making it possible for there to be ionic interactions with analytes. Other charged bases, such as amitripyline would be repelled by the charged particle surface, such that they exhibit less retention and fewer interactions with the particle surface. The peak shape of basic compounds can often be improved as a result of this Coulombic repulsion from the base particle.

However, under those same conditions, charged adds have the potential to undergo an anion- exchange mechanism with the DEAP ligands, which significantly increases their retention. The use of a counter-ion in the mobile phase, such as ammonium formate, could be used to attenuate the retention for acids.

In yet another aspect, a HPCM with a chromatographic surface produced by a diethylaminopropyl (DEAP) ionizable modifier, a C18 hydrophobic group and endcapping on a bridged ethylene hybrid particle has proven to be an exemplary embodiment for the separation of the acidic, polar molecules at pH > 10. At very high pH (pH > 10), the DEAP ligand would be relatively uncharged and interact as a polar neutral ligand, which may increase interactions of acidic, polar analytes. At the same time, the deprotonation of the DEAP ligand (pH>10) could be exploited to elute acidic analytes via the attenuation of the anion exchange mechanism.

In yet another aspects, a HPCM with a chromatographic surface produced by a diethylaminopropyl (DEAP) ionizable modifier, a C18 hydrophobic group and endcapping on a bridged ethylene hybrid particle has proven to be an exemplary embodiment for the separation of the acidic, polar molecules at relatively high pH conditions (7 < pH < 10). That the DEAP ligand has a high pKa makes it possible to achieve anionic retention at relatively high pH conditions (7 < pH < 10) that are favorable to online negative ion mode electrospray ionization (ESI) MS detection. In practice, separations of polar acids using the DEAP HPCM can be performed in numerous ways. Column temperatures between 30 and 90°C can be used. However, to balance optimizing diffusion coefficients and minimizing analyte degradation, it is preferred to use column temperatures between 40 and 80°C. Mobile phases can be generated by either binary or ternary systems, and in general, will entail composition changes that simultaneously reduce hydrophobic and anionic retention. For instance, an initial mobile phase composition of water (titrated to pH 8.5 with ammonium hydroxide) can be employed followed by a gradient change to a mobile phase comprised of 0.1 % ammonium hydroxide in 40:60 water/acetonitrile. In this embodiment of the invention, a gradient of increasing acetonitrile is applied to affect hydrophobic retention. Concomitantly, the pH of the mobile phase also shifts given the sizeable increase in ammonium hydroxide

concentration. The DEAP ligands become increasingly deprotonated across this sort of gradient, such that there is a weakening of the anionic retention mechanism. Combined with the acetonitrile change, a highly selective mixed mode separation is thereby achieved. This particular pH/solvent gradient technique is preferred as it can be performed with low ionic strength eluents, a feature advantageous for robust MS detection. On the other hand, in yet another embodiment, a gradient of increasing counter ion concentration could be employed along with an increase in acetonitrile concentration. Ammonium formate containing eluent of increasing and up to 200 mM concentrations could be used. Moreover, in other embodiments, organic solvents other than acetonitrile could be used, including both aprotic and protic solvents as well as those varying with respect to dielectric constant. These alternative solvents include but are not limited to methanol, ethanol, isopropanol, and tetrahydrofuran.

In other aspects, the acidic, polar molecule is eluted from the high purity

chromatographic material with weakly acidic mobile phases at 2.5 < pH < 7, including but not limited to mobile phases comprised of 0.01 to 0.5% formic acid, 1 to 50 mM ammonium formate and 1 to 50 mm ammonium acetate or combinations thereof. Elution can be initiated by either a gradient or isocratic separation. Elution may or may not entail a change in ionic strength and conductivity. In other aspects, the ionizable modifying reagent contains a pyridylethyl group or diethylaminopropyl (DEAP) group and elution of the adsorbed acidic, polar molecule from the high purity chromatographic material is performed with weakly acidic mobile phases at 2.5 < pH < 7, including but not limited to mobile phases comprised of 0.01 to 0.5% formic acid, 1 to 50 mM ammonium formate and 1 to 50 mm ammonium acetate or combinations thereof. Elution can be initiated by either a gradient or isocratic separation. Elution may or may not entail a change in ionic strength and conductivity.

In one embodiment, the HPCM of the invention does not have chromatographically enhancing pore geometry. In another embodiment, the HPCM of the invention has chromatographically enhancing pore geometry.

In certain embodiments, the HPCM of the invention has a surface area of about 25 to 1100 m ² /g; about 80 to 500 m ² /g; or about 120 to 330 m ² /g.

In other embodiments, the HPCM of the invention a pore volume of about 0.15 to 1.7 cm ² /g; or about 0.5 to 1.3 cm ² /g.

In certain other embodiments, the HPCM of the invention is non-porous.

In yet other embodiments, the HPCM of the invention has a micropore surface area of less than about 110 m ² /g; less than about 105 m ² /g; less than about 80 m ² /g; or less than about 50 m ² /g.

In still yet other embodiments, the HPCM of the invention has an average pore diameter of about 20 to 1500A; about 50 to ΙΟΟθΑ; about 100 to 75θΑ; or about 150 to 500A.

In another embodiment, the HPCM of the invention is hydrolytically stable at a pH of about 1 to about 14; at a pH of about 10 to about 14; or at a pH of about 1 to about 5.

In another aspect, the invention provides materials as described herein wherein the HPCM material further comprises a nanoparticle or a mixture of more than one nanoparticles dispersed within the chromatographic surface.

In certain embodiments, the nanoparticle is present in <20% by weight of the nanocomposite, <10% by weight of the nanocomposite, or <5% by weight of the

nanocomposite.

In other embodiments, the nanoparticle is crystalline or amorphous and may be silicon carbide, aluminum, diamond, cerium, carbon black, carbon nanotubes, zirconium, barium, cerium, cobalt, copper, europium, gadolinium, iron, nickel, samarium, silicon, silver, titanium, zinc, boron, oxides thereof, or a nitride thereof. In particular embodiments, the nanoparticle is a substance which comprises one or more moieties selected from the group consisting of nano-diamonds, silicon carbide, titanium dioxide, and cubic-boronitride.

In other embodiments, the nanoparticles may be less than or equal to 200 nm in diameter, less than or equal to 100 nm in diameter, less than or equal to 50 nm in diameter, or less than or equal to 20 nm in diameter.

Surface Modification

The HPCM materials of the invention may further be surface modified.

Thus, in one embodiment, the material as described herein may be surface modified with a surface modifier having the formula Z _a (R')bSi-R", where Z = CI, Br, I, Ci - C5 alkoxy, dialkylamino or trifluoromethanesulfonate; a and b are each an integer from 0 to 3 provided that a + b = 3; R' is a Ci - C6 straight, cyclic or branched alkyl group, and R" is a functionalizing group.

In another embodiment, the materials have been surface modified by coating with a polymer.

In certain embodiments, R' is selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl and cyclohexyl. In other embodiments, R' is selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, a cation or anion exchange group, an alkyl or aryl group containing an embedded polar functionality and a chiral moiety. In certain embodiments, R' is selected from the group consisting of aromatic, phenylalkyl, fluoroaromatic, phenylhexyl, pentafluorophenylalkyl and chiral moieties.

In one embodiment, R" is a Ci - C30 alkyl group. In a further embodiment, R" comprises a chiral moiety. In another further embodiment, R" is a Ci - C20 alkyl group.

In certain embodiments, the surface modifier comprises an embedded polar functionality. In certain embodiments, such embedded polar functionality includes carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, or triazole functionalities. In other

embodiments, such embedded polar functionality includes carbamate functionalities such as disclosed in U. S. Patent No. 5,374,755, and chiral moieties. Such groups include those of the general formula

wherein 1, m, o, r and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3 or 4 and q is an integer from 0 to 19; R-3 is selected from the group consisting of hydrogen, alkyl, cyano and phenyl; and Z, R', a and b are defined as above. Preferably, the carbamate functionality has the general structure indicated below:

O

— O-C-N— R5

H

5

wherein R may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl, tetradecyl, octadecyl, or benzyl. Advantageously, R^ is octyl, dodecyl, or octadecyl.

In certain embodiments, the surface modifier is selected from the group consisting of phenylhexyltrichlorosilane, pentafluorophenylpropyltrichlorosilane, octyltrichlorosilane, octadecyltrichlorosilane, octyldimethylchlorosilane and octadecyldimethylchlorosilane. In some embodiments, the surface modifier is selected from the group consisting of

octyltrichlorosilane and octadecyltrichlorosilane. In other embodiments, the surface modifier is selected from the group consisting of an isocyanate or Ι, -carbonyldiimidazole

(particularly when the hybrid group contains a (CH2)30H group).

In another embodiment, the material has been surface modified by a combination of organic group and silanol group modification.

In still another embodiment, the material has been surface modified by a combination of organic group modification and coating with a polymer. In a further embodiment, the organic group comprises a chiral moiety.

In yet another embodiment, the material has been surface modified by a combination of silanol group modification and coating with a polymer.

In other embodiments, the material has been surface modified via formation of an organic covalent bond between the particle's organic group and the modifying reagent. In still other embodiments, the material has been surface modified by a combination of organic group modification, silanol group modification and coating with a polymer.

In another embodiment, the material has been surface modified by silanol group modification.

In certain embodiments, the surface modified layer may be porous or non-porous.

Separation Devices and Kits and Methods of Use

Another aspect provides a variety of separations devices having a stationary phase comprising the HPCM materials as described herein. The separations devices include, e.g. , chromatographic columns, thin layer plates, filtration membranes, sample cleanup devices and microtiter plates.

The HPCM Materials impart to these devices improved lifetimes because of their improved stability. Thus, in a particular aspect, the invention provides a chromatographic column having improved lifetime, comprising

a) a column having a cylindrical interior for accepting a packing material, and b) a packed chromatographic bed comprising the high purity chromatographic material as described herein.

In another particular aspect, the invention provides a chromatographic device, comprising

a) an interior channel for accepting a packing material and

b) a packed chromatographic bed comprising the high purity chromatographic material as described herein.

The invention also provides for a kit comprising the HPCM materials as described herein, as described herein, and instructions for use. In one embodiment, the instructions are for use with a separations device, e.g., chromatographic columns, thin layer plates, filtration membranes, sample cleanup devices and microtiter plates. In another embodiment, the instructions are for the separation, isolation, purification, or detection of one or more acidic, polar molecules, e.g., organic acids, a-amino acids, phosphate sugars, nucleotides, other acidic, polar biologically relevant molecules.

The invention provides methods for selectively isolating/separating, purifying, detecting and/or analyzing an acidic, polar molecule or mixture of acidic, polar molecules using sthe HPCM materials as described herein. The methods of the invention are capable of separating and thereby resolving complex mixtures of compounds, allowing rapid isolation/separation, purification, detection and/or analysis of component compounds of such mixtures.

In one aspect the invention provides a method for selectively isolating an acidic, polar molecule from a sample, the method comprising the steps of:

a) loading a sample containing an acidic, polar molecule onto a chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety such that the acidic, polar molecule is selectively adsorbed onto the high purity chromatographic material; and b) eluting the adsorbed acidic, polar molecule from the high purity chromatographic material, thereby selectively isolating the acidic, polar molecule from the sample. In still another aspect, the invention provides a method for separating a plurality of acidic, polar molecules from a sample, the method comprising the steps of:

a) loading a sample containing a plurality of acidic, polar molecules onto

chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety such that the acidic, polar molecules are adsorbed onto the high purity chromatographic material; and b) eluting the adsorbed acidic, polar molecules from the high purity chromatographic material, thereby separating the acidic, polar molecules.

In yet another aspect, the invention provides a method for purifying an acidic, polar molecule contained in a sample, the method comprising the steps of:

a) loading a sample containing an acidic, polar molecule onto chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety such that the acidic, polar molecule are adsorbed onto the high purity chromatographic material; and b) eluting the adsorbed acidic, polar molecule from the high purity chromatographic material, thereby purifying an acidic, polar molecule.

In still yet another aspect, the invention provides a method for detecting an acidic, polar molecule in a sample, the method comprising the steps of:

a) loading a sample containing an acidic, polar molecule onto chromatographic separations device comprising a high purity chromatographic material comprising a chromatographic surface wherein the chromatographic surface comprises a hydrophobic surface group and one or more ionizable modifiers with the proviso that when the ionizable modifier does not contain a Zwitterion, the ionizable modifier does not contain a quaternary ammonium ion moiety such that the acidic, polar molecules are adsorbed onto the high purity chromatographic material; and b) eluting the adsorbed acidic, polar molecule from the high purity chromatographic material; and

c) detecting the acidic, polar molecule.

In certain aspects of the chromatographic methods of the invention, the acidic, polar molecule is selected from the group consisting of organic acids, a-amino acids, phosphate sugars, nucleotides, phosphonates, glyphosate, polar pesticides and other acidic, acidic, polar biologically relevant molecules.

Insofar as the target substance, i.e. , the acidic, polar molecule, is concerned, the methods of the invention work well on polar compounds, acidic compounds, basic compounds and any mixtures thereof. Thus, the acidic, polar molecules present in sample can be organic acids (e.g. , succinic acid, malic acid, cis aconitate acid, and nicotinic acid), a- amino acids (e.g., glutamine), phosphate sugars (e.g., glucose 6 phosphate, fructose 6 phosphate), nucleotides (e.g., adenosine mono-phosphate, nicotinic acid mono nucleotide, and adenosine diphosphate), phosphonates (etidronic acid), and other acidic, polar biologically relevant molecules (e.g., glufosinate, glyphosate, and aminomethylphosphonic acid), and mixtures thereof.

Synthesis of Materials of the Invention

The invention also provides methods for producing the high purity chromatographic materials (HPCM) materials described herein. In one embodiment, the invention provides a method for producing the HPCM described herein comprising the steps of: a. reacting a chromatographic core with an ionizable modifying reagent to obtain a ionizable modified material; and b. reacting the resultant material with a hydrophobic surface modifying group.

In another embodiment, the invention provides a method for producing the High purity chromatographic materials described herein comprising the steps of: a. reacting a chromatographic core with hydrophobic surface modifying group to obtain a surface modified material; and b. reacting the resultant material with an ionizable modifying reagent.

In another embodiment, the invention provides a method for producing the High purity chromatographic materials described herein comprising the steps of: a. reacting a chromatographic core with hydrophobic surface modifying group to obtain a surface modified material; and b. reacting the resultant material with an endcapping surface group, and c. reacting the resultant material with an ionizable modifying reagent.

In another embodiment, the invention provides a method for producing the High purity chromatographic materials described here comprising the steps of: a. reacting a chromatographic core with an ionizable modifying reagent to obtain an ionizable modified material; and b. reacting the resultant material to produce a hybrid surface layer; and c. reacting the resultant material with a hydrophobic surface modifying group.

In one aspect, the HPCM of the invention as described above is made with a charge ratio, B7A', from about 3 to about 133 wherein A' represents the ionizable modifier reagent charged in the preparation and B' represents the hydrophobic group charged in the preparation. In certain aspects, the charge ratio, B7A', is from about 4 to about 80, from about 4 to about 15, or from about 6 to about 7.

In one embodiment, the methods described herein further comprise the step of endcapping remaining silanol groups. In one embodiment, in the methods described the steps are performed simultaneously. In another embodiment, the pore structure of the as-prepared high purity

chromatographic materials us modified by hydrothermal treatment, which enlarges the openings of the pores as well as the pore diameters, as confirmed by nitrogen (N ₂ ) sorption analysis. The hydrothermal treatment is performed by preparing a slurry containing the as- prepared hybrid material and a solution of a base in water, heating the slurry in an autoclave at an elevated temperature, e.g., 100 to 200°C, for a period of 10 to 30 h. The use of an alkyl amine such as trimethylamine (TEA) or Tris(hydroxymethyl) methyl amine or the use of sodium hydroxide is advantageous. The thus-treated material is cooled, filtered and washed with water and methanol, then dried at 80°C under reduced pressure for 16 h.

In certain embodiments, following hydrothermal treatment, the surfaces of the high purity chromatographic materials are modified with various agents. Such "surface modifiers" include (typically) organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase. In certain aspects, when the HPCM is a hybrid material, it possesses possess both organic groups and silanol groups which may additionally be substituted or derivatized with a surface modifier.

The surface of the hydrothermally treated high purity chromatographic materials contains organic groups, which can be derivatized by reacting with a reagent that is reactive towards the materials' organic group. For example, vinyl groups on the material can be reacted with a variety of olefin reactive reagents such as bromine ( n), hydrogen (H2), free radicals, propagating polymer radical centers, dienes and the like. In another example, hydroxyl groups on the material can be reacted with a variety of alcohol reactive reagents such as isocyanates, carboxylic acids, carboxylic acid chlorides and reactive organosilanes as described below. Reactions of this type are well known in the literature, see, e.g., March, J. Advanced Organic Chemistry, 3 ^rd Edition, Wiley, New York, 1985; Odian, G. The Principles of Polymerization, 2 ^nd Edition, Wiley, New York, 1981.

In addition, the surface of the hydrothermally treated high purity chromatographic materials also contains silanol groups, which can be derivatized by reacting with a reactive organosilane. The surface derivatization of the high purity chromatographic materials is conducted according to standard methods, for example by reaction with

octadecyltrichlorosilane or octadecyldimethylchlorosilane in an organic solvent under reflux conditions. An organic solvent such as toluene is typically used for this reaction. An organic base such as pyridine or imidazole is added to the reaction mixture to catalyze the reaction. The product of this reaction is then washed with water, toluene and acetone. This material can be further treated by hydrolysis in a pH modified aqueous organic solution at ambient or elevated temperatures. An organic solvent such as acetone is typically used for this hydrolysis. Modification of pH can be achieved using acid or base modifiers, including trifluoroacetic acid, formic acid, hydrochloric acid, acetic acid, sodium or ammonium formate, sodium, potassium or ammonium acetate, phosphate buffers, ammonium hydroxide, ammonium carbonate, or ammonium bicarbonate. The product of the hydrolysis is then washed with water, toluene and acetone and dried at 80°C to 100°C under reduced pressure for 16 h. The resultant materials can be further reacted with a short-chain silane such as trimethylchlorosilane to endcap the remaining silanol groups, by using a similar procedure described above.

Surface modifiers such as disclosed herein are attached to the base material, e.g., via derivatization or coating and later crosslinking, imparting the chemical character of the surface modifier to the base material. In one embodiment, the organic groups of the high purity chromatographic materials react to form an organic covalent bond with a surface modifier. The modifiers can form an organic covalent bond to the materials organic group via a number of mechanisms well known in organic and polymer chemistry including but not limited to nucleophilic, electrophilic, cycloaddition, free-radical, carbene, nitrene and carbocation reactions. Organic covalent bonds are defined to involve the formation of a covalent bond between the common elements of organic chemistry including but not limited to hydrogen, boron, carbon, nitrogen, oxygen, silicon, phosphorus, sulfur and the halogens. In addition, carbon-silicon and carbon-oxygen-silicon bonds are defined as organic covalent bonds, whereas silicon-oxygen-silicon bonds that are not defined as organic covalent bonds.

The term "functionalizing group" includes organic functional groups which impart a certain chromatographic functionality to a chromatographic stationary phase, including, e.g., octadecyl (Cis) or phenyl. Such functionalizing groups are incorporated into base material directly, or present in, e.g., surface modifiers such as disclosed herein which are attached to the base material, e.g., via derivatization or coating and later crosslinking, imparting the chemical character of the surface modifier to the base material.

In certain embodiments, silanol groups are surface modified. In other embodiments, organic groups are surface modified. In still other embodiments, the high purity

chromatographic materials' organic groups and silanol groups are both surface modified or derivatized. In another embodiment, the high purity chromatographic materials are surface modified by coating with a polymer. In certain embodiments, surface modification by coating with a polymer is used in conjunction with silanol group modification, organic group modification, or both silanol and organic group modification. The ionizable modifier may be added to the material by silanol group modification, organic group modification, or by both silanol and organic group modification. The hydrophobic surface group may be added to the material by silanol group modification, organic group modification, or by both silanol and organic group modification.

More generally, the surface of high purity chromatographic materials may be modified by: treatment with surface modifiers including compounds of formula

Z _a (R') _D Si-R", where Z = CI, Br, I, Ci - C5 alkoxy, dialkylamino, e.g., dimethylamino, or trifluoromethanesulfonate; a and b are each an integer from 0 to 3 provided that a + b = 3; R' is a Ci - C6 straight, cyclic or branched alkyl group, and R" is a functionalizing group. In certain instances, such materials have been surface modified by coating with a polymer.

R' includes, e.g., methyl, ethyl, propyl, isopropyl, butyl, t-butyl, sec-butyl, pentyl, isopentyl, hexyl or cyclohexyl; preferably, R' is methyl.

The functionalizing group R" may include alkyl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, cation or anion exchange groups, an alkyl or aryl group containing an embedded polar functionalities or chiral moieties. Examples of suitable R" functionalizing groups include chiral moieties, C1-C30 alkyl, including C1-C20, such as octyl (Cs), octadecyl (Cis) and triacontyl (C30); alkaryl, e.g., Ci-C4-phenyl; cyanoalkyl groups, e.g., cyanopropyl; diol groups, e.g., propyldiol; amino groups, e.g., aminopropyl; and alkyl or aryl groups with embedded polar functionalities, e.g., .carbonate, amide, urea, ether, thioether, sulfinyl, sulfoxide, sulfonyl, thiourea, thiocarbonate, thiocarbamate, ethylene glycol, heterocyclic, and triazole functionalities or carbamate functionalities such as disclosed in U. S. Patent No. 5,374,755, and chiral moieties. In certain embodiments, R" is selected from the group consisting of aromatic, phenylalkyl, fluoroaromatic, phenylhexyl,

pentafluorophenylalkyl and chiral moieties. Such groups include those of the general formula

wherein 1, m, o, r and s are 0 or 1, n is 0, 1, 2 or 3 p is 0, 1, 2, 3 or 4 and q is an integer from 0 to 19; R-3 is selected from the group consisting of hydrogen, alkyl, cyano and phenyl; and Z, R', a and b are defined as above. Preferably, the carbamate functionality has the general structure indicated below:

wherein R may be, e.g., cyanoalkyl, t-butyl, butyl, octyl, dodecyl, tetradecyl, octadecyl, or benzyl. Advantageously, R ⁵ is octyl, dodecyl, or octadecyl.

In certain applications, such as chiral separations, the inclusion of a chiral moiety as a functionalizing group is particularly advantageous.

Polymer coatings are known in the literature and may be provided generally by polymerization or polycondensation of physisorbed monomers onto the surface without chemical bonding of the polymer layer to the support (type I), polymerization or

polycondensation of physisorbed monomers onto the surface with chemical bonding of the polymer layer to the support (type II), immobilization of physisorbed prepolymers to the support (type III) and chemisorption of presynthesized polymers onto the surface of the support (type IV). see, e.g., Hanson, et a , J. Chromat. A656 (1993) 369-380, the text of which is incorporated herein by reference. As noted above, coating the hybrid material with a polymer may be used in conjunction with various surface modifications described in the invention.

Thus, in certain embodiments, the hydrophobic surface modifier is selected from the group consisting of phenylhexyltrichlorosilane, pentafluorophenylpropyltrichlorosilane, octyltrichlorosilane, octadecyltrichlorosilane, octyldimethylchlorosilane and

octadecyldimethylchlorosilane. In a further embodiment, the surface modifier is selected from the group consisting of octyltrichlorosilane and octadecyltrichlorosilane. In another embodiment, the high purity chromatographic materials have been surface modified by a combination of organic group and silanol group modification.

In other embodiments, the high purity chromatographic materials have been surface modified by a combination of organic group modification and coating with a polymer.

In other embodiments, the high purity chromatographic materials have been surface modified by a combination of silanol group modification and coating with a polymer.

In another embodiment, the high purity chromatographic materials have been surface modified via formation of an organic covalent bond between the hybrid cores' and/or surrounding material materials' organic group and the modifying reagent.

In certain embodiments, the high purity chromatographic materials have been surface modified by a combination of organic group modification, silanol group modification and coating with a polymer.

In one embodiment, the high purity chromatographic materials have been surface modified by silanol group modification.

In another embodiment, the invention provides a method wherein the high purity chromatographic materials are modified by further including a porogen. In a further embodiment, the porogen is selected from the group consisting of cyclohexanol, toluene, mesitylene, 2-ethylhexanoic acid, dibutylphthalate, 1 -methyl-2-pyrrolidinone, 1-dodecanol and Triton X-45. In certain embodiments, the porogen is toluene or mesitylene.

In one embodiment, the invention provides a method wherein the high purity chromatographic materials are further modified by including a surfactant or stabilizer. In certain embodiments, the surfactant is Triton X-45, Triton XI 00, Triton X305, TLS, Pluronic F-87, Pluronic P-105, Pluronic P-123, sodium dodecylsulfate (SDS), ammonia

docecylsulfate, TRIS docecylsulfate, or Triton X-165. In certain embodiments, the surfactant is sodium dodecylsulfate (SDS), ammonia docecylsulfate, or TRIS docecylsulfate.

Certain embodiments of the synthesis of the HPCMs of the invention including hybrids, silica, particles, monoliths and superficially porous materials, are described above are further illustrated in the Examples below.

Examples

The present invention may be further illustrated by the following non-limiting examples describing the surface modification of porous chromatographic materials. Materials

All reagents were used as received unless otherwise noted. Those skilled in the art will recognize that equivalents of the following supplies and suppliers exist and, as such, the suppliers listed below are not to be construed as limiting.

Characterization

Those skilled in the art will recognize that equivalents of the following instruments and suppliers exist and, as such, the instruments listed below are not to be construed as limiting.

The %C, %H, %N values were measured by combustion analysis (CE-440 Elemental Analyzer; Exeter Analytical Inc., North Chelmsford, MA) or %C by Coulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet, IL). The specific surface areas (SSA), specific pore volumes (SPV) 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 SPV was the single point value determined for P/Po > 0.98 and the APD was calculated from the desorption leg of the isotherm using the BJH method. Scanning electron microscopic (SEM) image analyses were performed (JEOL JSM-5600 instrument, Tokyo, Japan) at 7 kV. Particle sizes were measured using a Beckman Coulter Multisizer 3 analyzer (30 μιτι aperture, 70,000 counts; Miami, FL). 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 /io ratio). Multinuclear ( ¹ C, ²⁹ Si) CP-MAS NMR spectra were obtained using a Bruker Instruments Avance-300 spectrometer (7 mm double broadband probe). The spinning speed was typically 5.0-6.5 kHz, recycle delay was 5 sec. and the cross-polarization contact time was 6 msec. Reported ¹ C and ²⁹ Si CP-MAS NMR spectral shifts were recorded relative to tetramethylsilane using the external standards adamantane ( ¹ C CP-MAS NMR, δ 38.55) and hexamethylcyclotrisiloxane ( ²⁹ Si CP-MAS NMR, δ -9.62). Populations of different silicon environments were evaluated by spectral deconvolution using DMFit software. [Massiot, D.; Fay on, F.; Capron, M.; King, I.; Le Calve, S.; Alonso, B.; Durand, J.-O.; Bujoli, B.; Gan, Z.; Hoatson, G. Magn. Reson. Chem. 2002, 40, 70-76] Titrations were performed using a Metrohm 716 DMS Titrino autotitrator with 6.0232.100 pH electrode (Metrohm, Hersau, Switzerland, or equivalent).

EXAMPLE 1

BEH porous hybrid particles (15 g, Waters Corporation, Milford, MA; 6.5 %C; SSA = 186 m ² /g; SPV = 0.79 cm /g; APD = 15 lA) of the formula (Oi.5SiCH ₂ CH ₂ SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) were refluxed in toluene (100 mL, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 1 hour. Reaction la used 7.2 g BEH material. Upon cooling the Component A silane additive was added, which included aminopropyltriethoxy silane (APTES, Gelest Inc., Morris ville, PA), 2-(2- (trichlorosilyl)ethyl)pyridine (2PE, Gelest Inc., Morrisville, PA), 2-(4- pyridylethyl)triethoxysilane (4PE, Gelest Inc., Morrisville, PA), N-trimethoxylsilylpropyl- Ν,Ν,Ν-trimethylammonium chloride (QPTMS, 50% solution in methanol, Gelest Inc., Morrisville, PA) or chloropropyltrimethoxysilane (CPTMS, Gelest Inc., Morrisville, PA). The reaction was heated to reflux for 1 hour. Upon cooling, imidazole (Aldrich, Milwaukee, WI) and octadecyldimethylchlorosilane (Component B, ODMCS, Aldrich or Gelest) were added. The reaction was then heated to reflux for 3 hours. For reactions lj and lk, 200 mL of toluene was used, and imidazole was added at the same time as the CPTMS. The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water and acetone (all solvents from Fisher Scientific). The product was then dried at 80 °C under reduced pressure for 16 hours. Reaction data is listed in Table 1. Product la was a control experiment that did not employ the use of a Component A silane additive. For products lb-11 the Component A silane additive charges ranged between 0.03-10.6 μιηοΐ/m ² and the charge molar ratio of Component B to A ranged from 0.19-66.6. Products lk and 11 introduced a chloropropyl silane group to the particle which is known to react with imidazole to obtain an imidazole propyl group [A. M. Lazarin, Y. Gushikem and S. C. deCastro, J. Mater. Chem., 2000, 10, 2526; B. Gadenne, P. Hesemann, J. J. E. Moreau Chem. Commun., 2004, 1768]. The reaction between the chloropropyl groups with imidazole was confirmed using ¹ C CP-MAS NMR spectroscopy.

The surface coverage of Component A silane additives was determined by the difference in particle %N after surface modification as measured by elemental analysis. As shown in Table 1, unbonded BEH particles as well as products la-lc did not have determinable nitrogen content by this measurement. ND stands for none determined. The surface coverage of Cis-groups was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis. Surface coverage of Cie- groups could be corrected by factoring out carbon content due to Component A silane additive by assuming complete condensation of the silane additive (correction method I), or by using the value obtained from the Component A silane additive coverage calculation (correction method II). For products lb-lj the correction in Cie coverage may be overestimated, but is still quite small (less than 0.11 μιηοΐ/ιη ² ). TABLE 1

EXAMPLE 2

Materials from Example 1 were modified with trimethylchlorosilane (TMCS, Gelest Inc., Morrisville, PA) using imidazole (Aldrich, Milwaukee, WI) in refluxing toluene (100 mL) for 4 hours. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker) and then dried at 80 °C under reduced pressure for 16 hours. Reaction data are listed in Table 2.

TABLE 2

EXAMPLE 3

BEH porous hybrid particles (Waters Corporation, Milford, MA; 6.5%C; SSA = 182-185 m ² /g; SPV = 0.72-0.76 cm ³ /g; APD = 142-151 A) of the formula

(Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) were refluxed in toluene (5 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean- Stark trap for 1 hour. Upon cooling the Component A silane additive was added, which included aminopropyltriethoxysilane (APTES, Gelest Inc., Morrisville, PA) 2-(4- pyridylethyl)triethoxy silane (4PE, Gelest Inc., Morrisville, PA), or

diethylphosphatoethyltriethoxysilane (DEPS, Gelest Inc. Morrisville, PA) or 2-(4- chlorosulfonylphenyl)ethyltrichlorosilane (SPETCS, 50% in toluene, Gelest Inc.,

Morrisville PA). The reaction was heated to reflux for 1 hour. Upon cooling, imidazole (Aldrich, Milwaukee, WI) and octadecyltrichlorosilane (Component B, ODTCS, Aldrich, Milwaukee, WI) were added. The reaction was then heated to reflux for 16 hours. Product 3c was reacted for 3 hours. Products 3af-3aj did not add a component B.

The reaction was then cooled and the product was filtered and was washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from J.T. Baker). The material was then refluxed in a acetone / aqueous 0.12 M ammonium acetate solution (Sigma Chemical Co., St. Louis, MO) for 2 hours (hydrolysis-type A), acetone/aqueous 0.1 M ammonium bicarbonate (pH 8) solution for 20 hours at 50°C (hydrolysis-type B), or acetone/aqueous 0.1 M ammonium bicarbonate (pH 10) solution for 20 hours at 50°C

(hydrolysis-type C). The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from J.T. Baker). The product was then dried at 80 °C under reduced pressure for 16 hours. Reaction data is listed in Table 3. The silane additive (Component A) charges ranged from 0.03- 3.70 μπιοΐ/ιη ² and the molar ratio of charge molar ratio of Component B to A ranged from 4.3-133.4.

The surface coverage of Cis-groups was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis. Correction for coverage of Cis-groups, obtained by factoring out carbon content due to silane additive by assuming complete condensation of the silane additive, were small for this dataset (less than 0.15 μπιοΐ/ιη ² ) and were not included in Table 3. Product 3aj had an ion-exchange capacity of 0.22 mequiv/g by titration. TABLE 3

EXAMPLE 4

Materials from Example 3 were modified with triethylchlorosilane (TECS, Gelest Inc., Morrisville, PA) or tert-butyldimethylchlorosilane (TBDMCS, Gelest Inc., Morrisville, PA) using imidazole (Aldrich, Milwaukee, WI) in refluxing toluene (5 mL/g) for 4-20 hours. The reaction was cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker) and then dried at 80 °C under reduced pressure for 16 hours. Reactions 4a-4g and 4m-4ab were reacted for 4 hours, reactions 4h-41 were reacted for 20 hours. Additional

trimethylchlorosilane (TMCS, Gelest Inc., Morrisville, PA) and imidazole was added to reactions 4m-4ab and the reaction was heated for an additional 16 hours. Selected products were further reacted with TMCS (reaction 4k) or hexamethyldisilazane (reaction 4c, Gelest Inc., Morrisville, PA) in a similar process. Reaction data are listed in Table 4.

TABLE 4

Particles Silane Imidazole

Product Precursor (g) Silane (g) (g) %C

4a 3a 15 TECS 4.18 2.27 17.32

4b 3b 15 TECS 4.18 2.27 14.53

4c 3c 50 TECS 13.95 7.75 17.58

4d 3d 10 TECS 2.79 1.51 17.27

4e 3d 10 TBDMCS 2.79 1.51 16.99

4f 3e 32 TECS 9.00 4.85 16.96

4g 3f 32 TECS 8.4.0 4.55 17.10

4h 3g 15 TBDMCS 4.18 2.27 14.54

4i 3h 15 TBDMCS 4.18 2.27 17.44

4j 3i 15 TBDMCS 4.18 2.27 14.62

4k 3j 20 TBDMCS 5.48 2.97 14.61

41 3k 15 TBDMCS 4.18 2.27 17.30

4m 31 15 TECS 2.06 1.12 15.14

4n 3m 15 TECS 2.06 1.12 14.82 4o 3n 20 TECS 2.74 1.49 14.28

4p 3o 20 TECS 2.74 1.49 15.43

4q 3p 20 TECS 2.74 1.49 15.26

4r 3q 20 TECS 2.74 1.49 15.36

4s 3r 20 TECS 2.74 1.49 15.61

4t 3s 20 TECS 2.74 1.49 15.67

4u 3t 20 TECS 2.74 1.49 14.96

4v 3u 20 TECS 2.38 1.29 15.92

4w 3v 20 TECS 2.24 1.22 14.99

4x 3w 20 TECS 2.21 1.20 15.97

4y 3x 20 TECS 2.25 1.22 14.97

4z 3ac 20 TECS 2.78 1.50 15.71

4aa 3 ad 10 TECS 2.76 1.50 15.52

4ab 3ae 22 TECS 2.70 1.47 15.51

4ac 3 ad 10 TBDMCS 3.40 3.00 15.36

EXAMPLE 5

BEH porous hybrid particles (Waters Corporation, Milford, MA; 3.9 μιη, 6.68 %C; SSA = 182 m ² /g; SPV = 0.75 cm ³ /g; APD = 148 A) of the formula

(Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent

6,686,035) were refluxed in toluene (5 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean- Stark trap for 1 hour. Upon cooling the Component A silane additive was added, which included aminopropyltriethoxysilane (APTES, Gelest Inc., Morrisville, PA), 2-(4- pyridylethyl)triethoxy silane (4PE, Gelest Inc., Morrisville, PA), or 2- (carbomethoxy)ethyltrichlorosilane (CMETCS, Gelest Inc., Morrisville, PA). The reaction was heated to reflux for 1 hour. For reactions 5f and 5g a mixture of APTES and CMETCS were used. Upon cooling, imidazole (Aldrich, Milwaukee, WI) or diisopropyl ethylamine (DIPEA, Aldrich, Milwaukee, WI) and the Component B silane was added, which included phenylhexyltrichlorosilane (PTCS), octyltrichlorosilane (OTCS, Aldrich, Milwaukee, WI), pentafluorophenylpropyltrichlorosilane (PFPPTCS), or octadecyldimethylchlorosilane (ODMCS, Aldrich, Milwaukee, WI). Products 5a-5h used imidazole. Products 5i-5t used DIPEA. The reaction was then heated to reflux for 16 hours.

The reaction was cooled and the product was filtered and was washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from J.T. Baker). The material was then hydrolyzed as detailed in Example 3. Products 5a-5h used hydrolysis type A. Products 5i-5u used hydrolysis type C. The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from J.T. Baker). The product was then dried at 70 °C under reduced pressure for 16 hours. Reaction data is listed in Table 5. The Component A silane additive charges ranged from 0.03-0.35 μπιοΐ/ιη ² and the charge molar ratio of Component B to A ranged from 6.5-133.3. The surface coverage was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis.

TABLE 5

EXAMPLE 6

Material from Example 5 was modified with triethylchlorosilane (TECS, Gelest Inc., Morrisville, PA) or tert-butyldimethylchlorosilane (TBDMCS, Gelest Inc., Morrisville, PA) using imidazole (Aldrich, Milwaukee, WI) in refluxing toluene (5 mL/g) for 17-20 hours. Additional TMCS and imidazole was added to reactions 6e, 6i-6o, and 6q after 4 hours and the reaction was heated for an additional 16 hours. For reactions 6i and 6j diisopropyl ethylamine (DIPEA, Aldrich, Milwaukee, WI) was used in place of imidazole. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker) and then dried at 70 °C under reduced pressure for 16 hours. Samples of product 6p were further hydrolyzed in an aqueous acetonitrile solution or hydrolysis C in Example 3. No noticeable change in carbon content was observed. Reaction data are listed in Table 6.

TABLE 6

Particles Silane Base

Product Precursor (g) Silane (g) (g) %C

6a 5a 15 TBDMCS 4.11 2.23 11.36

6b 5b 15 TBDMCS 4.11 2.23 13.44

6c 5c 15 TBDMCS 4.11 2.23 11.71

6d 5d 15 TBDMCS 4.11 2.23 13.33

6e 5e 15 TECS 2.06 1.12 11.56

6f 5f 15 TBDMCS 4.11 2.23 16.66

6g 5g 15 TBDMCS 4.11 2.23 16.76

6h 5h 15 TBDMCS 4.11 2.23 16.89

6i 51 20 TECS 2.57 2.64 11.55

6j 5n 19 TECS 2.59 2.66 12.90

6k 5o 40 TECS 5.31 2.88 13.60

61 5p 10 TECS 1.45 0.79 13.91

6m 5q 67 TECS 9.20 5.00 13.75 6n 5r 70 TECS 9.20 5.00 13.06

6o 5s 20 TECS 2.92 1.58 13.80

6p 5s 40 TBDMCS 9.21 4.99 13.32

6q 5u 36 TECS 5.00 2.70 13.43

EXAMPLE 7

BEH porous hybrid particles (15 g, 1.7 μιη, Waters Corporation, Milford, MA; 6.5%C; SSA = 92 m ² /g; SPV = 0.73 cmVg; APD = 311 A) of the formula

(Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) were refluxed in toluene (100 mL, Fisher Scientific, Fairlawn, NJ) using a Dean- Stark trap for 2 hours. Upon cooling the Component A silane additive

aminopropyltriethoxysilane (0.018 g, 0.06 μπιοΐ/ιη ² charge, Gelest Inc., Morrisville, PA) was added and the reaction was heated to reflux for 1 hour. Upon cooling, imidazole (5.06 g, Aldrich, Milwaukee, WI) and the Component B silane tert-butyldimethylchlorosilane (2.08 g, Gelest Inc., Morrisville, PA) were added. The reaction was then heated to reflux for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker) and then dried at 80 °C under reduced pressure for 16 hours. The surface coverage of product 7a, determined by the difference in particle %C before and after the surface modification (7.88 %C) as measured by elemental analysis, was determined to be

2.50 μιηοΐ/m ² . EXAMPLE 8

Porous silica particles (Waters Corporation, Milford, MA; 3.5 μπι; SSA = 251 m ² /g; SPV = 0.80 cm ³ /g; APD = 119 A) were refluxed in toluene (5 mL per gram of silica, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 1 hour. Upon cooling the Component A silane additive was added, which included aminopropyltriethoxysilane (APTES, Gelest Inc., Morrisville, PA) or 2-(4-pyridylethyl)triethoxysilane (4PE, Gelest Inc., Morrisville, PA). Product 8a used APTES. Products 8b-d used 4PE. The reaction was heated to reflux for 1 hour. Upon cooling, imidazole (Aldrich, Milwaukee, WI) or diisopropyl ethylamine (DIPEA, Aldrich, Milwaukee, WI) and the Component B silane was added, which included phenylhexyltrichlorosilane (PTCS), pentafluorophenylpropyltrichlorosilane (PFPPTCS), or octadecyldimethylchlorosilane (ODMCS, Aldrich, Milwaukee, WI). Products 8a and 8b used Imidazole. Products 8c and 8d used DIPEA. The reaction was then heated to reflux for 20 hours. The reaction was then cooled and the product was filtered and was washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from J.T. Baker). The material was then refluxed in an acetone / aqueous 0.1 M ammonium acetate solution (Sigma Chemical Co., St. Louis, MO) for 3.5 hours. Products 8b, 8c and 8d were heated at 50 °C for 20 hours. The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from J.T. Baker). The product was then dried at 80 °C under reduced pressure for 16 hours. The surface coverage of the product, determined by the difference in particle %C before and after the surface modification as measured by elemental analysis. Products 8a, was further reacted in a similar manner as described for product 4c, to yield products 8e. Products 8b and 8d were further reacted in a similar manner as described for product 4m, to yield products 8f and 8g. Reaction data are listed in Table 7.

EXAMPLE 9 Samples of porous particles from Example 2, 4, 6, and 8 were used for the separation of a mixture of neutral, polar and basic compounds listed in Table 8. The 2.1x100 mm chromatographic columns were packed using a slurry packing technique. The chromatographic system consisted of an ACQUITY UPLC ^® System and an ACQUITY UPLC ^® Tunable UV detector. Empower 2 Chromatography Data Software (Build 2154) was used for data collection and analysis. Mobile phase conditions were: 20 mM

K2HPO4/KH2PO4, pH 7.00±0.02 / methanol (40/60 v/v); flow rate: 0.25 mL/min;

temperature: 30 °C; detection: 254 nm; analytes: uracil, propranolol, butylparaben, naphthalene, dipropylphthalate, acenaphthene, and amitriptyline. Columns 4g and 8a were tested at 23°C. It can be seen that columns packed with particles from Examples 2, 4, 6, 7 and 8 provide sufficient retention and resolution in the separation of neutral, polar, and basic compounds under these conditions. Relative retention is the retention time of the analyte divided by the retention time of acenaphthene. Therefore values less than one, indicate less retention than acenaphthene, and values greater than one, indicate more retention than acenaphthene. (Relative retention is a well known parameter in the field of HPLC.)

TABLE 7

TABLE 8

Retention Relative Retention:

Product Factor: Propranolol / Butylparaben / Naphthalene / Dipropyl phthalate / Amitriptyline /

Acenaphthene Acenaphthene Acenaphthene Acenaphthene Acenaphthene Acenaphthene

2a 8.45 0.218 0.300 0.458 0.540 1.768

2b 8.79 0.218 0.291 0.455 0.520 1.860

4f 13.49 0.145 0.210 0.422 0.382 1.578

4g 13.80 0.140 0.219 0.431 0.382 1.508

4j 8.87 0.232 0.287 0.458 0.505 2.254

4k 8.14 0.215 0.292 0.459 0.482 1.624

4m

4n

4o

4p

4q

4r

4s

4t

4u

4z

4aa

4ac

6e

6f

6g

6h

8a

Commercial

<2 μm Hybrid

Ci8 Column

Commercial

<2 μηι Silica

Ci8 Column

Commercial

<2 μm Silica 17.85 0.153 0.194 0.417 0.378 1.404 Ci8 Column

Commercial

<2 μm Silica 6.70 2.663 0.284 0.480 0.495 17.912 Ci8 Column

EXAMPLE 10

Samples of porous particles from Example 2, 4, 6, and 8 were evaluated for USP peak tailing factors using the mobile phase and test conditions of Example 9. The results are shown in Table 9. Peak tailing factors is a well known parameter in the field of HPLC 5 (a lower value corresponds to reduced tailing). It is evident that columns packed with

particles from Examples 2, 4, 6, 7 and 8 have comparable tailing factors to commercially available Cis-columns.

TABLE 9

Tailing Factor for:

Product Dipropyl-

Propranolol Butylparaben Naphthalene Acenaphthene Amitriptyline phthalate

2a LOO 1.42 1.54 1.26 1.25 1.37

2b 1.86 1.30 1.24 1.26 1.15 1.98

4f 1.03 1.37 1.28 1.33 1.26 1.88

4g 0.95 1.31 1.25 1.28 1.22 1.91

4j 1.51 1.19 1.17 1.16 1.17 3.44

4k 1.32 1.16 1.16 1.14 1.20 1.45

4m 1.25 1.28 1.29 1.28 1.26 1.55

4n 1 67 1.12 1.11 1.09 1.06 1.41

4o 1.18 1.16 1.16 1.13 1.11 1.31

4p 1.79 1.18 1.18 1.15 1.13 1.91

4q 1.57 1.15 1.17 1.14 1.16 1.60

4r 1.52 1.17 1.17 1.15 1.15 2.39

4s 1.09 1.41 1.27 1.29 1.14 1.14

4t 1.27 1.41 1.23 1.32 1.13 1.31

4u 1.37 1.16 1.18 1.16 1.17 2.19

4z 1.22 1.39 1.45 1.42 1.31 2.44

4aa 1.61 1.25 1.30 1.24 1.22 2.42

4ac 1.50 1.31 1.51 1.40 1.50 2.58

6e 1.34 1.24 1.25 1.23 1.29 1.47 6f 1.96 1.23 1.29 1.26 1.31 2.66

6g 1.92 1.24 1.28 1.25 1.29 2.69

6h 1.92 1.22 1.27 1.25 1.29 2.81

8a 1.06 1.11 1.08 1.11 1.08 2.76

Commercial

<2 μηι

0.88 1.34 1.24 1.29 1.14 1.15 Hybrid Cis

Column

Commercial

<2 μιη

0.96 1.17 1. 10 1.33 1.10 6.95 Silica Cis

Column

Commercial

<2 μπι

0.95 1.35 1.22 1.32 1.10 1.77 Silica Cis

Column

Commercial

<2 μηι

4.19 1.34 1.29 1.28 1.12 1.34 Silica Cis

Column

EXAMPLE 11

Samples of porous particles from Example 2-8 were used for the separation of a mixture of neutral, polar and basic compounds listed in Table 10. The 2.1x100 mm

5 chromatographic columns were packed using a slurry packing technique. Columns packed with products 5i-5m, 6i-6o, and 8c-8g used 2.1x50 mm chromatographic columns. The chromatographic system consisted of an ACQUITY UPLC ^® System and an ACQUITY UPLC ^® Tunable UV detector. Empower 2 Chromatography Data Software (Build 2154) was used for data collection and analysis. Mobile phase conditions were: 15.4 mM

10 ammonium formate, pH 3.00±0.02 / acetonitrile (65/35 v/v); flow rate: 0.25 mL/min; temperature: 30 °C; detection: 254 nm; analytes: uracil, pyrenesulfonic acid, desipramine, amitriptyline, butylparaben, and toluene. Columns 4g and 8a were tested at 23 °C.

It can be seen that columns packed with particles from Examples 2-8 provide sufficient retention and resolution in the separation of neutral, polar, and basic compounds under these conditions. Relative retention is the retention time of the analyte divided by the retention time of toluene. Therefore values less than one, indicate less retention than toluene, and values greater than one, indicate more retention than toluene (relative retention is a well known parameter in the field of HPLC).

TABLE 10

Relative Retention:

Retention Pyrenesulfonic Desipramine/ Amitriptyline/ Butylparaben/

Factor: acid/ Toluene Toluene Toluene

Product Toluene Toluene

2a 10.95 0.148 0.275 0.373 0.965

2b 11.73 0.669 0.180 0.244 0.975

3ac 10.31 1.913 0.151 0.215 1.073

3 ad 9.70 2.137 0.138 0.197 1.076

4j 11.37 0.491 0.184 0.250 1.081

4k 10.82 0.216 0.231 0.311 1.101

4m 10.75 0.227 0.219 0.297 1.115

4n 10.76 0.245 0.216 0.291 1.125

4o 10.31 0.219 0.234 0.316 1.098

4p 11.07 0.177 0.242 0.328 1.074

4q 9.69 0.354 0.188 0.250 1.211

4r 11.31 0.154 0.243 0.329 1.001

4s 11.73 0.180 0.251 0.339 1.087

4t 11.76 0.199 0.237 0.319 1.099

4u 11.34 0.229 0.240 0.326 1.140

4z 12.78 0.208 0.233 0.316 1.044 1.051

1.026

1.151

1.179

1.114

1.171

1.163

1.218

0.865

0.854

0.849

1.119

1.156

1.111

1.081

1.147

1.138

1.105

1.258

0.808

1.129

0.997

1.112

0.894

0.924 Column

Commercial

<2 μιη

1.57 1.030 2.882 3.934 10.172

Silica Ci8

Column

Commercial

<2 μm

9.61 0.170 0.595 0.888 1.076

Silica Ci8

Column

EXAMPLE 12

Samples of porous particles from Example 2-8 were evaluated for USP peak tailing factors using the mobile phase and test conditions of Example 1 1. The results are shown in Table 11. Peak tailing factor is a well known parameter in the field of HPLC (a lower value corresponds to reduced tailing). It is evident that columns packed with particles from Examples 2-8 provide have comparable tailing factors to commercially available Cis- columns.

TABLE 11

Tailing Factor for:

Product Pyrenesulfonic Desipramine Amitriptyline Butylparaben Toluene acid

2a 24.51 1.81 2.21 1.06 1.03

2b 4.60 1.63 1.81 1.00 1.01

3ac 1.89 2.16 2.32 1.06 1.02

3 ad 1.17 1.69 1.66 1.03 1.01

4j 1.86 1.65 2.06 1.04 1.03

4k 1.68 1.63 1.95 1.06 1.01

4m 1.60 1.45 1.54 1.14 1.05

4n 1.58 1.36 1.51 1.00 0.98 o 1.43 1.46 1.70 1.04 1.01p 1.76 1.54 1.81 1.05 1.01q 1.25 1.40 1.61 1.03 0.99r 1.72 1.69 2.04 1.05 1.04s 1.67 1.90 2.51 1.03 1.02t 1.75 1.82 2.38 1.02 1.00u 1.78 2.05 2.56 1.05 1.00z 2.18 2.84 3.26 1.08 1.05aa 1.90 1.80 2.03 1.05 1.05ac 2.24 1.90 2.04 1.09 1.07i 1.46 1.54 1.49 1.08 0.97j 1.27 1.68 1.61 1.09 1.12k 1.50 1.38 1.36 1.12 1.051 1.65 1.48 1.47 1.36 1.23m 1.19 1.21 1.17 1.01 1.01e 1.37 1.30 1.35 1.09 1.05f 2.30 1.50 1.66 1.12 1.07g 2.83 1.61 1.79 1.11 1.06h 2.35 1.77 2.05 1.10 1.07i 2.60 1.36 1.38 1.08 1.12j 1.51 1.40 1.39 1.08 1.10k 1.88 1.64 1.64 1.08 0.851 1.49 1.24 1.29 0.99 0.83m 1.39 1.26 1.29 1.08 1.09n 1.46 1.30 1.32 1.08 1.04o 1.53 1.44 1.43 1.08 1.07a 1.67 1.54 1.57 1.18 1.15a 3.40 1.49 1.62 1.04 1.05c 1.30 1.17 1.16 1.00 0.92f 1.55 1.44 1.57 1.06 1.05 8g 1.61 1.42 1.45 1.10 1.08

Commercial

<2 μιη

1.71 2.76 3.32 1.03 1.03

Hybrid Cis

Column

Commercial

<2 μm

1.40 2.81 3.58 1.01 1.02

Silica Ci8

Column

Commercial

<2 μm

1.75 3.20 3.82 1.04

Silica Ci8

Column

Commercial

<2 μιη

1.65 2.20 2.65 1.06 1.01

Silica Ci8

Column

EXAMPLE 13

Samples of porous particles from Example 2, 4-8 were used for the separation of a mixture of neutral and basic compounds listed in Table 12. The 2.1x 50 mm

chromatographic columns were packed using a slurry packing technique. The

chromatographic system consisted of an ACQUITY UPLC® System and an ACQUITY UPLC® Tunable UV detector. Empower Chromatography Data Software (Build 1 154) was used for data collection and analysis. Gradient conditions: 15-65% acetonitrile (solvent B) over 4.6 minutes in 0.1% formic acid (Solvent A) followed by a 1.4 minute hold; flow rate: 0.4 mL/min; temperature: 30 °C; detection: 260 nm; basic test mix prepared in 16.7% methanol: uracil, metoprolol tartrate, papaverine, amitriptyline; neutral test mix prepared in 16.7% methanol: uracil, prednisone, caffeine. Columns packed with products 51, 5n, 6i, 6j, 8c and 8f used 15-95%) acetonitrile. Comparison Column A and B were commercially available and contained 2.7 μιη Cis-bonded superficially porous silica packing material. Comparison Column C was commercially available and contained 1.7 μπι porous hybrid particles of the formula (Oi.sSiCHiCLhSiOi.sXSiChX that was surface modified with ODTCS followed by endcapping.

Peak capacities were calculated using the average of the peak widths (4□) over three injections. The determination of peak capacity and the problems caused by poor peak shape and resulting poor peak capacities for basic analytes in low pH gradient separations is well known in the field of HPLC and UPLC. By comparing the ratio of peak capacities for a basic analyte (amitriptyline) to a neutral analyte (prednisone) under these test conditions, a better comparison of basic analyte chromatographic performance can be made. A peak capacity ratio near one indicates similar performance of the basic and neutral analytes. A peak capacity ratio less than 0.8 indicates a substantial decrease in

chromatographic performance. A peak capacity greater than one indicates an improvement in chromatographic performance for the basic analytes over the neutral analyte.

Differences due to changes in particle size can be observed by comparing the peak capacity ratios for columns packed with products 4u, 4j, and 4n. While these products are of similar Component A and B type and charges, they range in particle size from 1.8 μπι (product 4u), 2.9 μπι (product 4j) and 3.9 μπι (product 4n). The peak capacity ratios were determined to be 0.86, 1.09 and 1.02, respectively. We can conclude that the particle size impacts performance under these conditions, especially for < 2 μπι packing materials. Product 4u still has significant improvements in peak capacity ratios over Comparison Columns A-D.

The impact of Component A silane additive can be observed by comparing the peak capacity ratios for columns packed with product 2a and 2d. These products are of similar size, Component B silane type and Component B silane charge. Product 2a does not contain a Component A silane additive. Product 2d was prepared with APTES charged at 0.3 μπιοΐ/ιη ² . The peak capacity ratios were determined to be 0.72 and 1.18,

respectively. We can conclude that Component A silane additive type improves performance under these conditions.

Differences in Component A silane additive type can be observed by comparing the peak capacity ratios for columns packed with products 4c and 4i. These products are of similar size and Component B silane charge. While they were prepared using the same Component A silane additive charge, the Component A silane additive type was APTES for product 4c and 4PE for product 4i. The peak capacity ratios were determined to be 0.74 and 0.38, respectively. We conclude that Component A silane additive type impacts performance under these conditions.

Differences in Component A silane additive charge can be observed by comparing the peak capacity ratios for columns packed with products 4h and 4j . These products are of similar size, Component B silane type and Component B silane charge. While these products were prepared with the same Component A silane additive type, the Component A charge varied from 0.06 μιηοΐ/ιη ² (product 4h) to 0.3 μιηοΐ/ιη ² (product 4j). The peak capacity ratios were determined to be 0.67 and 1.09, respectively. We conclude that Component A silane additive charge impacts performance under these conditions.

Differences in Component B silane type can be observed by comparing the peak capacity ratios for columns packed with product 4k and 6e. These products are of similar size, Component A silane additive type and Component A silane additive charge. While these products were prepared with the same Component B silane charge, the Component B silane type was ODTCS for product 4k and OTCS for product 6e. The peak capacity ratios were determined to be 1.02 and 0.34, respectively. We conclude the Component B silane type impacts performance under these conditions.

Differences in Component B silane charge can be observed by comparing the peak capacity ratios for columns packed with products 41 and 4j . These products are of similar size, Component A silane additive type and charge. While these products were prepared with the same Component B silane, the Component B silane charge was 4 μιηοΐ/ιη ² (product 41) and 2 μιηοΐ/ιη ² (product 4j). The peak capacity ratios were determined to be 0.45 and 1.09, respectively. We conclude the Component B silane charge impacts performance under these conditions. TABLE 12

A B

Amitnptyline Prednisone Ratio

Product Pc Pc A/B

2a 95 132 0.72

2d 99 84 1.18

4c 88 119 0.74

4h 66 98 0.67

4i 33 88 0.38

4j 109 100 1.09

4k 88 86 1.02

41 49 109 0.45

4m 97 96 1.01

4n 86 84 1.02

4o 169 155 1.09

4p 150 148 1.01

4q 144 149 0.96

4r 147 157 0.94

4s 176 181 0.97

4t 181 177 1.02

4u 202 235 0.86

4z 169 209 0.81

4aa 162 163 0.99

4ac 179 154 1.16

5i 237 219 1.08

5j 231 218 1.06

5k 160 147 1.09

51 165 144 1.15

5m 126 116 1.09

5n 171 137 1.25 Attorney Docket No.: 8185.0163WO

EXAMPLE 14

Samples of porous particles from Example 2 and 4 were evaluated for efficiency difference upon increased loading of basic analytes. The 4.6 x 150 mm chromatographic columns were packed using a slurry packing technique. The chromatographic system consisted of an Alliance HPLC ^® System and a Waters 996 PDA detector. Empower 2 Chromatography Data Software (Build 2154) was used for data collection and analysis; injection volume 20 μΐ _^ ; flow rate: 1.0 mL/min; temperature: 30 °C; detection: 230 nm; analytes: amitriptyline or propranolol (prepared 60 μ§/ιηΙ. in mobile phase). Loading range on Table 13 : 0.1 μg-2.5 μg analyte on column. In order to have comparable retention factors (0.9-2.0), mobile phase conditions were modified for separations using amitriptyline [0.05% TFA in acetonitrile/water (60/40 v/v)] and propranolol [0.05% TFA in acetonitrile/water (70/30 v/v)]. Comparison column A was commercially and contained 5 μπι Ci8-bonded porous silica packing material. Comparison column B was commercially available and contained 5 μπι porous hybrid packing of the formula

(Oi. ₅ SiCH2CH2SiOi. ₅ )(Si02)4, that was surface modified with ODTCS followed by endcapping. Comparison columns C and D were commercially available and contained 5 μπι porous silica packing that was surface modified with an organofunctional silane followed by Cis surface modification.

The observation of decreased efficiency and worsening of peak shape for basic analytes at increased loadings when used under low pH isocratic conditions is well known in the field of HPLC and UPLC. Not limited to theory, this worsening of separation performance for basic analytes has been attributed with analyte overloading. As tabulated in Table 12, the decreased performance at increased loadings is determined as the percent loss in column efficiency between 0.1-1.2 μg or 0.1-2.5 μg loading of amitriptyline or propranolol.

Similar results were obtained for amitriptyline and propranolol at the 1.2 μg and

2.5 μg loadings. Columns that performed well on this test, including columns containing products 2c, 2g, and 4g, had a low loss in efficiency (<20%) at the 1.2 μg analyte loading. These columns had comparable performance to Comparison Columns A and C and improved performance over Comparison Columns B and D. These well-performing columns had a further decrease in efficiency between 1.2 μg and 2.5 μg loadings of approximately 100%. Other columns tested had a greater loss in efficiency (>20%) at 1.2 μg analyte loading, as well as a further decrease in efficiency between 1.2 μg and 2.5 μg loadings of approximately 25-50%).

The impact of Component A silane additive type can be observed by comparing the loss in amitriptyline efficiency (1.2 μg on column) for columns packed with product 2c and 2g. These products have the same Component B silane type and Component B silane charge. While they were prepared using the same Component A silane additive charge, the Component A silane additive was APTES for product 2c and 4PE for product 2g. The losses in amitriptyline efficiency were determined to be 4% and 13%, respectively.

The impact of Component A silane charge can be observed by comparing the loss in amitriptyline efficiency (1.2 μg on column) for columns packed with product 4c, 4f, and 4g. These products have the same Component B silane type and Component B silane charge. While they were prepared using the same Component A silane additive type, the Component A silane charge was 0.06 μιηοΐ/m ² for product 4c, 0.12 μιηοΐ/m ² for product 4f and 0.20 μιηοΐ/m ² for product 4g. The losses in amitriptyline efficiency were determined to be 40%, 34% and 10%, respectively.

The impact of Component B silane type can be observed by comparing the loss in amitriptyline efficiency (1.2 μg on column) for columns packed with product 2c and 4b. These products have the same Component A silane additive type and Component A silane additive charge. While they were prepared using the same Component B silane charge, the Component B silane type was ODMCS for product 2c and ODTCS for product 4b. The losses in amitriptyline efficiency were determined to be 4% and 43%, respectively.

TABLE 13

EXAMPLE 15

The general procedure for modifying surface silanol groups to result in the display of hydrophobic surface group and ionizable modifier that is detailed in Examples 1, 3, 5, 7 and 8 is applied to modify the surface silanol groups of different porous materials.

Included in this are monolithic, spherical, granular, superficially porous and irregular materials that are silica, hybrid inorganic/organic materials, hybrid inorganic/organic surface layers on hybrid inorganic/organic, silica, titania, alumina, zirconia, polymeric or carbon materials, and silica surface layers on hybrid inorganic/organic, silica, titania, alumina, zirconia or polymeric or carbon materials. The particles size for spherical, granular or irregular materials vary from 5-500 μιη; more preferably 15-100 μιη; more preferably 20-80 μιη; more preferably 40-60 μιη. The APD for these materials vary from 30 to 2,000 A; more preferably 40 to 200 A; more preferably 50 to 150 A. The SSA for these materials vary from 20 to 1000 m ² /g; more preferably 90 to 800 m ² /g; more preferably 150 to 600 m ² /g; more preferably 300 to 550 m ² /g. The TPV for these materials vary from 0.3 to 1.5 cm ³ /g; more preferably 0.5 to 1.2 cmVg; more preferably 0.7 to 1.1 cm ³ /g. The macropore diameter for monolithic materials vary from 0.1 to 30 μπι, more preferably 0.5 to 25 μπι, more preferably 1 to 20 μιη.

The ionizable modifier, component A, is selected from groups used in Examples 1, 3, 5, 7 and 8 or is selected from a group having formula (I), formula (II) or formula (III) including an acidic ionizable modifier including, but not limited to, protected and unprotected versions of alkyl, aryl, and arylalkyl groups containing phosphoric, carboxylic, sulfonic, and boronic acids

Preferred silane ionizable modifying reagents of formula I and II include 4-pyridyl alkyl trialkoxysilane, 3-pyridyl alkyl trialkoxysilane, 2-pyridyl alkyl trialkoxysilane, imidazole alkyl trialkoxysilane, aminoalkyl trialkoxysilane, and mono- and di- alkylaminoalkyl trialkoxysilane.

Preferred silane ionizable modifying reagents of formula III include the trisilanol, trialkoxysilane or trichlorosilane, the protected and deprotected acid forms, chloro forms, as well as salts of sulfonic acid alkyl silanes, sulfonic acid phenylalkyl silanes, sulfonic acid benzylalkyl silanes, sulfonic acid phenyl silanes, sulfonic acid benzyl silanes, carboxylic acid alkyl silanes, carboxylic acid phenylalkyl silanes, carboxylic acid benzylalkyl silanes, carboxylic acid phenyl silanes, carboxylic acid benzyl silanes, phosphoric acid alkyl silanes, phosphonic acid phenylalkyl silanes, phosphonic acid benzylalkyl silanes, phosphonic acid phenyl silanes, phosphonic acid benzyl silanes, boronic acid alkyl silanes, boronic acid phenylalkyl silanes, boronic acid benzylalkyl silanes, boronic acid phenyl silanes, boronic acid benzyl silanes. EXAMPLE 16

Residual silanol groups from select materials prepared in Example 15 are further reacted following protocols detailed in Examples 2, 4, and 6. EXAMPLE 17

In a general procedure propanol hybrid surrounded hybrid particles (product 17a) were prepared in a multistep procedure as follows;

Acetoxypropyltrimethoxysilane (700 g, Gelest Inc., Morrisville, PA) was mixed with ethanol (374 g, anhydrous, J.T. Baker, Phillipsburgh, NJ) and an aqueous solution of 0.01 M Acetic Acid (22 g, J.T. Baker, Phillipsburgh, NJ) in a flask. The resulting solution was agitated and refluxed for 16 hours in an atmosphere of argon or nitrogen. Alcohol was removed from the flask by distillation at atmospheric pressure. Residual alcohol and volatile species were removed by heating at 110° C for 17 hours in a sweeping stream of argon or nitrogen. The resulting polyorganoalkoxy siloxanes was a clear viscous liquid had a viscosity of 95 cP.

This polyorganoalkoxy siloxanes was added to a suspension of BEH porous hybrid particles (20 g, Waters Corporation, Milford, MA; 6.5%C; SSA = 190 m ² /g; SPV = 0.80 cm ³ /g; APD = 155 A) of the formula (prepared following the method described in U.S. Patent 6,686,035) in dry toluene (Fisher Scientific, Fairlawn, NJ; 5 mL/g). This reaction was heated at 80 °C for one hour and 110 °C for 20 hours using a Dean-Stark trap to remove residual water. The reaction was cooled to room temperature and particles were isolated on 0.5 μπι filtration paper and washed repeatedly using ethanol (anhydrous, J.T.Baker, Phillipsburgh, NJ). The material was then heated to 50 °C in a suspension with ethanol (3 mL/g, anhydrous, J.T.Baker, Phillipsburgh, NJ), deionized water (7 mL/g) and 30% ammonium hydroxide (20 g; J.T.Baker, Phillipsburgh, NJ) for 4 hours. The reaction was then cooled and the product was filtered and washed successively with water and methanol (Fisher Scientific, Fairlawn, NJ). The product was then dried at 80 °C under reduced pressure for 16 hours.

The particles were then mixed with an aqueous solution of 0.3 M

tris(hydroxymethyl)aminomethane (TRIS, Aldrich Chemical, Milwaukee, WI) at a slurry concentration of 5 mL/g. The pH of the resultant slurry was adjusted to 9.8 using acetic acid (J.T. Baker, Phillip sburgh, NJ). The slurry was then enclosed in a stainless steel autoclave and heated to 155 °C for 20 hours. After cooling the autoclave to room temperature, the product was were isolated on 0.5 μιη filtration paper and washed with water and methanol (Fisher Scientific, Suwanee, GA). The particles were then dried at 80 °C under vacuum for 16 hours.

The particles were then dispersed in a 1 molar hydrochloric acid solution (Aldrich, Milwaukee, WI) for 20 h at 98 °C. The particles were isolated on 0.5 μπι filtration paper and washed with water to a neutral pH, followed by acetone (HPLC grade, Fisher

Scientific, Fairlawn, N.J.). The particles were dried at 80 °C under vacuum for 16 h.

Products obtained by this approach have 8.1-8.6 %C; SSA = 150-166 m ² /g; SPV = 0.6- 0.7 cm ³ /g; APD = 134-145 A). Structural analysis was performed using MR

spectroscopy. Surface coverage of propanol groups, determined by the difference in particle %C using elemental analysis, was 3.2-3.8 μπιοΐ/ιη ² .

EXAMPLE 18

Propanol hybrid surrounded hybrid particles from Example 17 were modified with octadecyl isocyanate (ODIC, Aldrich Chemical), pentafluorophenyl isocyanate (PFPIC, Aldrich Chemical), 2,2-Diphenylethyl isocyanate (DPEIC, Aldrich Chemical), 4- cyanophenyl isocyanate (4CPIC, Aldrich Chemical), or 3-cyanophenyl isocyanate (3CPIC, Aldrich Chemical) in dry toluene (5 mL/g, J.T.Baker) under an argon blanket. The suspension was heated to reflux (110 °C) for 16 h and then cooled to <30°C. The particles were transferred to a filter apparatus and washed exhaustively with toluene and acetone. The material was then treated as detailed in the hydrolysis section of Example 3, or the material was heated for an hour at 50 °C in a 1 : 1 v/v mixture of acetone and 1% trifluoroacetic acid (Aldrich, Milwaukee, WI ) solution (10 mL/g particles) (Hydrolysis D). The reaction was then cooled and the product was filtered and washed successively with acetone and toluene (heated at 70 °C). The product was then dried at 70 °C under reduced pressure for 16 hours. Reaction data is listed in Table 14. The surface coverage of carbamate groups was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis.

TABLE 14

Component B

Carbamate

Isocyanate Isocyanate Surface dp Particles mass Charge Hydrolysis Coverage

Product (μηι) (g) Isocyanate (g) (μηιοΐ/ηι ² ) Type %C (μηιοΐ/m ² )

18a 3.0 25 ODIC 11.9 10.0 B 15.93 2.55

18b 3.0 60 ODIC 28.9 10.0 B 15.86 2.47

18c 3.0 40 ODIC 19.3 10.0 B 15.28 2.26

18d 3.0 40 ODIC 19.3 10.0 B 15.28 2.26

18e 4.0 50 ODIC 27.3 11.8 B 15.49 2.47

18f 3.5 25 ODIC 12.0 10.0 B 14.58 2.07

18g 3.5 25 ODIC 6.0 5.0 B 13.04 1.52

18h 3.5 15 ODIC 7.2 10.0 B 14.3 2.00

18i 3.5 15 ODIC 7.2 10.0 B 14.55 2.09

18j 3.5 15 ODIC 7.2 10.0 B 14.55 2.09

18k 3.5 10 ODIC 4.8 10.0 B 13.64 1.76

181 3.5 10 ODIC 4.8 10.0 B 13.78 1.81

18m 3.5 10 ODIC 4.8 10.0 B 13.87 1.85

18n 4.9 10 ODIC 3.6 8.0 B 12.97 1.58

18o 4.9 33 ODIC 12.4 8.0 B 14.45 2.12

18p 3.5 50 ODIC 18.9 8.0 B 14.82 2.19

18q 3.5 50 ODIC 18.9 8.0 B 14.78 2.18

18r 3.5 50 ODIC 18.9 8.0 B 14.95 2.24

18s 3.5 60 ODIC 23.6 8.0 B 15.10 2.20

18t 4.9 60 ODIC 22.6 8.0 B 15.24 2.35

18u 3.0 20 PFPIC 6.7 10.0 D 12.24 3.98 18v 3.5 12 DPEIC 4.3 10.0 C 14.05 2.36

18w 3.0 45 4CPIC 10.4 10.0 D 13.01 3.63

18x 4.9 40 4CPIC 3.1 3.45 C 11.88 2.90

18y 4.9 40 3CPIC 3.1 3.45 C 11.96 2.97

EXAMPLE 19

The materials of Example 18 were further modified aminopropyltriethoxysilane (APTES, Gelest Inc., Morrisville, PA), 2-(4-pyridylethyl)triethoxysilane (4PE, Gelest Inc., Morrisville, PA) or 2-(2-pyridylethyl)trimethoxysilane (2PE, Gelest Inc., Morrisville, PA), in refluxing toluene (5 mL/g) for 20 hours. Products 19a and 19b were reacted for 4 hours. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker). The material was then treated as detailed in the hydrolysis section of Example 3, or the material was heated for an hour at 50 °C in a 1 : 1 v/v mixture of acetone and 1% trifluoroacetic acid (Aldrich, Milwaukee, WI ) solution (10 mL/g particles) (Hydrolysis D). The reaction was then cooled and the product was filtered and washed successively water, toluene, 1 : 1 v/v acetone/water and acetone and then dried at 70 °C under reduced pressure for 16 hours. Reaction data is listed in Table 15.

TABLE 15

Cc imponen t A

Silane Charge

Silane Additive Molar

Particles Silane mass Charge Ratio Hydrolysis

Product Precursor (g) Additive (g) (μηιοΐ/ηι ² ) B/A Type %C

19a 18a 10 APTES 0.01 0.03 333 none 16.82

19b 18a 10 APTES 0.02 0.05 200 none 16.82

19c 18b 30 4PE 0.04 0.03 333 none 15.79

19d 18b 15 4PE 0.04 0.06 167 A 15.81

19e 18b 20 4PE 0.04 0.05 200 B 15.82

19f 18b 30 4PE 0.08 0.06 167 none 15.79 9g 18b 17 4PE 0.08 0.11 91 A 15.78 9h 18b 20 4PE 0.08 0.09 111 B 15.79

19i 18b 30 4PE 0.04 0.03 333 B 15.79

19j 18b 30 4PE 0.08 0.06 167 A 15.79 9k 18c 30 4PE 0.08 0.06 167 B 15.18

191 18d 30 4PE 0.08 0.06 167 A 15.229m 18d 30 4PE 0.08 0.06 167 D 15.18 9n 18d 10 4PE 0.01 0.03 439 B 15.17 9o 18e 10 4PE 0.25 0.59 20 B 15.00 9p 18e 10 APTES 0.01 0.04 295 B 15.34 9q 18e 10 APTES 0.004 0.01 1180 B 15.32

19r 18f 25 2PE 0.04 0.04 250 B 14.73

19s 18g 24 2PE 0.04 0.04 125 B 13.32

19t 18h 10 4PE 0.09 0.20 50 C 14.20 9u 18i 10 4PE 0.13 0.30 33 C 14.36 9v 18k 9 4PE 0.11 0.30 33 C 13.789w 181 8 4PE 0.04 0.10 100 C 13.63 9x 18m 9 4PE 0.19 0.50 20 C 13.87 9y 18n 9 2PE 0.10 0.30 27 C 13.27 9z 18o 30 4PE 0.39 0.30 27 C 14.419aa 18p 30 4PE 0.39 0.30 27 C 14.829ab 18q 30 4PE 0.39 0.30 27 C 14.799ac 18r 30 4PE 0.39 0.30 27 C 14.819ad 18s 55 4PE 0.74 0.30 27 C 14.879ae 18t 30 4PE 0.39 0.30 27 C 14.629af 18u 6 4PE 0.01 0.03 333 none 11.699ag 18v 9 4PE 0.12 0.31 32 C 14.079ah 18w 8 4PE 0.01 0.03 347 none 12.779ai 18x 10 4PE 0.21 0.51 7 C 11.839aj 18x 10 4PE 0.12 0.29 12 C 11.80 19ak 18y 10 4PE 0.21 0.51 7 C 11.93

19al 18y 10 4PE 0.12 0.29 12 C 11.87

EXAMPLE 20

Selected materials of Example 19 were further modified by endcapping as detailed in Example 4. Data is listed in Table 16.

TABLE 16

EXAMPLE 21

Propanol hybrid surrounded hybrid particles from Example 17 were modified with 2-(4-pyridylethyl)triethoxysilane (4PE, Gelest Inc., Morrisville, PA) in refluxing toluene (5 mL/g) for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker). The material was then treated as hydrolysis C of Example 3. The reaction was then cooled and the product was filtered and washed successively water, toluene, 1 : 1 v/v acetone/water and acetone. Selected products were then dried at 70 °C under reduced pressure for 16 hours. Reaction data is listed in Table 17.

TABLE 17

EXAMPLE 22

Products from Example 21 were modified with isocyanate as detailed in Example 18 using hydrolysis C. Reaction data is listed in Table 18. TABLE 18

EXAMPLE 23

The concentration of surface pyridyl groups (ionizable modifier) were quantified for select materials prepared in Examples 3 , 4, 21 and 22 using the following procedure. 2-(4-pyridylethyl)triethoxysilane (1.12 μηιοΐ, Gelest Inc., Morrisville, PA) in methanol (0.4 mL, HPLC grade) was added to a sample (0.2000 g) from Example 3, 21 or 22. The sample was then digested using sodium hydroxide solution (4.0 mL, 2.5 M) at 64 °C for 60 minutes. The sample was filtered through a Millex-LCR filter (0.45 μηι, 25 mm,

Millipore) and was extracted with hexane (HPLC grade). The aqueous layer was then analyzed using a UV/Visible spectrophotometer (300-240 nM, 0.1 nM interval, scan speed = 120 nM/min, slit width = 2 nM). The concentration of pyridyl groups were calculated using the absorbance at two wavelengths with corrections made for base particle contribution to absorbance. The results are listed in Table 19. These results indicate a reduced concentration of pyridylethyl groups (component A) on the surface than was charged. Using the determined coverage of component B we can determine the determined surface coverage ratio of B/A. The result of this is a larger range of surface coverage ratio of B/A (8-190) than molar charge ratio (6-80).

TABLE 19

Component Component B Component Component

A A B

Product Ionizable Hydrophobic Charge Ionizable Hydrophobic Surface

Modifier Group Molar Modifier Group Coverage

Charge Charge Ratio Coverage Coverage Ratio

(μηιοΙ/Γη ² ) (μηιοΙ/Γη ² ) B/A (μηιοΙ/Γη ² ) (μηιοΙ/Γη ² ) B/A

3 ah 3.70 — — 0.890 — —

4v 0.35 2.53 7 0.160 2.45 15

4w 0.35 2.07 6 0.250 2.09 8

4x 0.25 2.53 10 0.140 2.51 18

4y 0.25 2.07 8 0.210 2.05 8

8b 0.30 2.3 8 0.28 2.53 9

8f 0.30 2.3 8 0.27 2.53 9

21a 0.10 — — 0.020 — —

21b 0.20 — — 0.029 — —

21c 0.10 — — 0.020 — —

21d 0.20 — — 0.025 — —

21e 0.20 — — 0.017 — —

21f 0.20 — — 0.013 — —

21g 0.20 — — 0.01 1 — —

21h 0.20 — — 0.019 — —

21i 0.20 — — 0.031 — —

22a 0.10 8.00 80 0.019 2.21 1 16

22b 0.20 8.00 40 0.028 2.23 80

22c 0.10 8.00 80 0.018 2.20 122

22d 0.20 8.00 40 0.022 2.24 102

22e 0.20 8.00 40 0.015 2.10 140

22f 0.20 8.00 40 0.01 1 2.09 190

22g 0.20 8.00 40 0.017 2.05 121 22h 0.20 8.00 40 0.029 2.01 69

22i 0.30 8.00 27 0.038 1.96 52

22j 0.15 8.00 53 0.028 1.98 71

22k 0.15 8.00 53 0.030 1.96 65

221 0.15 8.00 53 0.025 1.96 78

EXAMPLE 24

To a suspension of 5 μηι BEH porous hybrid particles (25 g, Waters Corporation, Milford, MA; 6.5%C; SSA = 190 m ² /g; SPV = 0.80 cm ³ /g; APD = 155 A) of the formula (Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) in dry toluene (250 mL, Fisher Scientific) was added Component A, 2-(4- pyridylethyl)triethoxysilane (0.2182 g, 0.2 μπιοΐ/ιη ² , Gelest Inc., Morrisville, PA), before following the general procedure for preparing propanol hybrid surrounded hybrid particles detailed in Example 17. Product 24a of this reaction incorporated a low level of ionizable modifier during the formation of the propanol hybrid surrounded hybrid particles having 8.3 %C and 3.70 μπιοΐ/ιη ² propanol groups.

EXAMPLE 25

A portion product 24a (16.2 g) from Example 24 was reacted with Component B, octadecyl isocyanate (7.51 g, 10 μπιοΐ/ιη ² ), in a similar process detailed in Example 18, using hydrolysis C. The product of this reaction had 14.27 %C and 2.00 μπιοΐ/ιη ² carbamate groups. This resulting product 25a had a molar charge ratio of B/A of 50.

EXAMPLE 26

The general procedure to prepare a propanol hybrid surrounded core material, detailed in Examples 17 are applied to different porous materials. Included in this are core materials detailed in Example 15. EXAMPLE 27

Modification of the surface of these propanol hybrid surrounded core materials prepared in Example 26 with a component B hydrophobic group is accomplished using silane approaches detailed in Examples 1, 3 or 5 or with isocyanate approaches detailed in Examples 18.

Further modification of the surface of these materials with a component A ionizable modifier is accomplished using silane approached detailed in Examples 19. Alternatively the surface propanol groups is reacted with ionizable modifying reagents of formula type I, or II where Z is isocyanate or 1 -carbamoyl imidazole, following the approach detailed in Example 18. Preferred ionizable modifiers include 4-pyridyl alkylisocyanates, 3-pyridyl alkylisocyanates, 2-pyridyl alkylisocyanates, imidazole alkylisocyanates, l-(N-(4-pyridyl alkyl)carbamoyl)imidazole, l-(N-(3-pyridyl alkyl)carbamoyl)imidazole, l-(N-(2-pyridyl alkyl)carbamoyl)imidazole, and 1 -(N-(imidazol- 1 -yl-alkyl)carbamoyl)imidazole.

Alternatively the surface propanol groups is reacted with ionizable modifying reagents of formula III where Z is isocyanate or 1 -carbamoyl imidazole, following the approach detailed in Example 18. Preferred ionizable modifiers include acid-protected and acid-non-protected versions of isocyanato-alkyl sulfonic acid, isocyanato-alkyl carboxylic acid, isocyanato-alkyl phosphoric acid, isocyanato-alkyl boronic acid, [(imidazole- 1- carbonyl)-amino]-alkyl sulfonic acid, [(imidazole-l-carbonyl)-amino]-alkyl carboxylic acid, [(imidazole- l-carbonyl)-amino]-alkyl phosphoric acid, [(imidazole- 1-carbonyl)- amino]-alkyl boronic acid, isocyanato-aryl sulfonic acid, isocyanato-aryl carboxylic acid, isocyanato-aryl phosphoric acid, isocyanato-aryl boronic acid, [(imidazole- 1-carbonyl)- amino]-aryl sulfonic acid, [(imidazole- l-carbonyl)-amino]-aryl carboxylic acid,

[(imidazole- l-carbonyl)-amino]-aryl phosphoric acid, [(imidazole- l-carbonyl)-amino]-aryl boronic acid, isocyanato-aryl alkyl sulfonic acid, isocyanato-aryl alkyl carboxylic acid, isocyanato-aryl alkyl phosphoric acid, isocyanato-aryl alkyl boronic acid, [(imidazole- 1- carbonyl)-amino]-aryl alkyl sulfonic acid, [(imidazole- l-carbonyl)-amino]-aryl alkyl carboxylic acid, [(imidazole- l-carbonyl)-amino]-aryl alkyl phosphoric acid, [(imidazole- 1- carbonyl)-amino]-aryl alkyl boronic acid, isocyanato-alkyl aryl alkyl sulfonic acid, isocyanato-alkyl aryl alkyl carboxylic acid, isocyanato-alkyl aryl alkyl phosphoric acid, isocyanato-alkyl aryl alkyl boronic acid, [(imidazole-l-carbonyl)-amino]-alkyl aryl alkyl Attorney Docket No.: 8185.0163WO sulfonic acid, [(imidazole- l-carbonyl)-amino]-alkyl aryl alkyl carboxylic acid,

[(imidazole-l-carbonyl)-amino]-alkyl aryl alkyl phosphoric acid, and [(imidazole- 1- carbonyl)-amino]-alkyl aryl alkyl boronic acid. EXAMPLE 28

Modification of the surface of these propanol hybrid surrounded core materials as detailed in Example 27, but the ionizable group is reacted before the hydrophobic group.

EXAMPLE 29

The general procedure to prepare a propanol hybrid surrounded core material having an ionizable group, detailed in Examples 24 is applied to different core materials. Included in this are core materials detailed in Example 15. Modification of the surface of these propanol hybrid surrounded core materials with a hydrophobic group is

accomplished using silane approaches detailed in Examples 1, 3 or 5 or is accomplished using isocyanate approaches detailed in Examples 18.

EXAMPLE 30

Further modification of the surface of materials prepared in Examples 27-29 is accomplished using approaches detailed in Examples 2, 4, 6, and 20 or surface propanol groups are future reacted with alkyl isocyanate or aryl isocyanates as detailed in Exampl 18.

EXAMPLE 31

The general approach to prepare a hybrid surrounded hybrid particle is used to prepare new hybrid surrounded materials that have reactive surface groups other than silanols and propanol groups, following a general approach detailed in Example 17 and 24, using core materials detailed in Example 15. When hybrid surfaces are prepared that have vinyl, haloalkyl, aminoalkyl, epoxy or phenyl groups, different reactions are performed to attach the hydrophobic or ionizable modifier. Vinyl groups are modified using radical addition, metathesis, epoxidation and hydrosilylation. Haloalkyl groups are modified by nucleophillic displacement and Grinard reactions. Aminoalkyl groups are reacted with acids, isocyanates or nucleophillic displacement. Epoxy groups are hydrolyzed to present surface alcohol groups, or reactions with amines. Phenyl groups are substituted with chloromethyl, sulfonic or nitro groups. lonizable modifying reagents of formula type I, II or III result where Z represents a chemically reactive group, including (but not limited to) silane, silanol, ether, amine, alkylamine, dialkylamine, isocyanate, acyl chloride, triflate, isocyanate, thiocyanate, imidazole carbonate, 1 -carbamoyl imidazole, HS-ester, carboxylic acid, ester, epoxide, alkyne, alkene, azide, -Br, -CI, or -I.

Further modifications of these materials is accomplished as detailed in Examples

27-30.

EXAMPLE 32

In a general procedure propanol surrounded particles containing an ionizable modifier are prepared in a multistep procedure. Products 3af-3ah from Example 3 are reacted with acetoxypropyltrichlorosilane in dry toluene using imidazole. The reaction is heated to reflux for 20 hours before cooling, filtering, and washing with toluene, 1 : 1 v/v acetone/water, and acetone. The material is refluxed acetone/aqueous 0.1 M ammonium bicarbonate (pH 10) solution for 20 hours at 50°C. The reaction is cooled and the product is filtered and is washed successively with toluene, 1 : 1 v/v acetone/water, and acetone. The product is then hydrolyzed in 1 N HC1 for 20 hours at an elevated temperature. The reaction is cooled and the product is filter and is washed with water and acetone. The product is dried at 80 °C under reduced pressure for 16 hours. Products prepared by this approach have surface pyridylethyl and propanol groups.

EXAMPLE 33

The general procedure to prepare a propanol hybrid surrounded core material using acetoxypropyltrichlorosilane or a polyorganoalkoxy siloxane, having an initial

modification with ionizable modifier is applied to different core materials. Included in this are core materials detailed in Example 15. The modification of these core materials with an ionizable modifier is accomplished using silane approaches detailed in Examples 1, 3 or 5, or is accomplished using ionizable modifying reagents of formula I, II or III detailed in Example 15. The general approach to modify core materials with

acetoxypropyltrichlorosilane is detailed in Example 33. The general approach to modify core materials with acetoxypropyltrichlorosilane is detailed in Example 17.

EXAMPLE 34

Acetoxypropyltrimethoxysilane (323 g, Gelest Inc., Morrisville, PA) was mixed with 2-(4-pyridylethyl)triethoxysilane (13.04 g, Gelest Inc., Morrisville, PA), ethanol (218 g, anhydrous, J.T. Baker, Phillipsburgh, NJ) and an aqueous solution of 2.2 M Acetic Acid (26 g, J.T. Baker, Phillipsburgh, NJ) in a flask. The resulting solution was agitated and refluxed for 16 hours in an atmosphere of argon or nitrogen. Alcohol was removed from the flask by distillation at atmospheric pressure. Residual alcohol and volatile species were removed by heating at 110 °C for 5 hours in a sweeping stream of argon or nitrogen. The resulting polyorganoalkoxy siloxane, Product 34a, was a clear viscous liquid had a viscosity of 27 cP.

EXAMPLE 35

In a general procedure, propanol hybrid surrounded core materials containing an ionizable modifier are prepared by a multistep procedure where Product 34a from Example 34 is used in place of the polyorganoalkoxy siloxane in Example 17.

Alternatively this general procedure to prepare add the ionizable modifier before the preparation of the propanol hybrid surrounded core material is applied to different core materials. Included in this are core materials detailed in Example 15.

EXAMPLE 36

Modification of the surface of materials prepared in Examples 31-33 and 35 with a hydrophobic group is accomplished using silane approaches detailed in Examples 1, 3 or 5 or with isocyanate approaches detailed in Examples 18. EXAMPLE 37

Secondary surface modification of materials prepared in Examples 36 is accomplished using approaches detailed in Examples 2, 4, 6, and 20 or with isocyanate approaches detailed in Examples 18

EXAMPLE 38

Products prepared in Examples 15, 16, 19-22, 24-25, 27-33, and 35-37 are chromatographically evaluated as detailed in Examples 9-14. Concentration of ionizable modifier are determined as detailed in Example 23.

EXAMPLE 39

Samples of porous particles from Product 4aa and a 3 μπι commercially available Ci8 column were evaluated for changes in retention of ionized analytes when exposed to mobile phases of different pH. The 2.1 x 50 mm chromatographic columns were packed using a slurry packing technique. The chromatographic system consisted of an ACQUITY UPLC ^® System and an ACQUITY UPLC ^® Tunable UV detector. Empower

Chromatography Data Software (Build 1154) was used for data collection and analysis; injection volume 2 μΐ.; flow rate: 0.8 mL/min; temperature: 30 °C; detection: 260 nm; analytes: metoprolol and amitriptyline. Data were compared before (initial) and after (final) 7 cycles; each cycle included alternately 7 injections in a 0.1 % formic

acid/acetonitrile gradient followed by 17 injections in a 10 mM ammonium bicarbonate (pH 10)/acetonitrile gradient. Both acidic and pH 10 gradients ran from 5 to 95 % acetonitrile in 2.5 minutes.

As shown in Figure 1, changes in retention of ionized analytes when exposed to mobile phases of different pH is a problem that is known in the art. The commercially available Cis column experienced a 7% change in retention for amitriptyline, while Product 4aa experienced a 0.4% change in retention for amitriptyline under these conditions. While not limited to theory, it has been proposed that slow surface

equilibration is to blame. Because conventional high-purity reversed-phase columns have much reduced surface charge at low pH, very small changes in surface charge may cause a large change in retention for ionized analytes. This effect is exacerbated by the use of low- ionic- strength mobile phases. The change in selectivity is not due to loss of bonded phase because the change is reversible, and no loss of retention is observed for neutral analytes. Storage and/or equilibration of columns in the low-pH mobile phase (allowing time for diffusion) will eventually return them to their original selectivity. This slow equilibration does not occur at elevated pH because of the relatively high concentration of deprotonated silanols.

These data indicate that, unlike the commercially available Cis column, Product 4aa can be used in method development screens of high and low pH gradient conditions with the assurance that the method will work on an unused column.

EXAMPLE 40

Similar to Example 13, samples of porous particles from Example 4j and a commercially available Cis column were used for the separation of a mixture of neutral and basic compounds. The basic test mix prepared included uracil, metoprolol tartate, labetalol, amitriptyline and the neutral test mix included uracil, prednisone, caffeine. The comparison Cis column was commercially available and contained 3.5 μιη porous hybrid particles of the formula that was surface modified with ODTCS followed by endcapping.

As shown in Figure 2, the results for Product 4j has drastic improvements in peak shape for basic analytes under these conditions, compared to the comparison Cis column that did not have any ionizable modifier added. This great improvement can also be demonstrated in improved peak capacities, as detailed in Example 13.

EXAMPLE 41

Samples of porous particles from Example 2 were evaluated for isocratic loading behavior for amitriptyline. The 4.6 x 150 mm chromatographic columns were packed using a slurry packing technique. The chromatographic system consisted of an Alliance HPLC ^® System and a Waters 996 PDA detector. Empower 2 Chromatography Data Software (Build 2154) was used for data collection and analysis; injection volume 20 μΐ.; flow rate: 1.0 mL/min; temperature: 30 °C; detection: 230 nm; analyte: amitriptyline (prepared 60 μ§/ιηΙ. in mobile phase) loading range: 0.3 μg - 1.2 μg analyte on column; mobile phase: 0.05% TFA in 40 % acetonitrile.

Deterioration of peak shape of basic analytes with increasing loading concentration is a well known problem for separations performed on UPCM at low pH. The effect of surface charge on peak profiles can be observed, as shown in Figure 3, by comparing the change in peak profiles with increasing analyte concentration for Products 2b, 2d, and 2e. Product 2e has a high level of ionizable modifier shows fronting/ Anti-Langmuirian peak shape suggesting a concave Langmuirian isotherm; (b) Product 2d has an optimal level of ionizable modifier shows nearly symmetrical Gaussian/linear peak shape suggesting a linear Langmuirian isotherm; (c) Product 2b has a very low level of ionizable modifier shows tailing/Bi-Langmuirian peak shape suggesting a convex Langmuirian isotherm. The importance of maintaining good peak shape with increased analyte loading is well known in the art. Product 2d has an optimized surface charge to give high efficiencies for loads that far exceed those attainable on ordinary reversed-phase columns.

EXAMPLE 42

Samples of porous particles from Product 4aa and a 3 μπι commercially available Ci8 column were evaluated for isocratic loading behavior for amitriptyline. The 2.1 x 50 mm chromatographic columns were packed using a slurry packing technique. The chromatographic system consisted of an ACQUITY UPLC ^® System and an ACQUITY UPLC ^® Tunable UV detector. Empower Chromatography Data Software (Build 1154) was used for data collection and analysis; injection volume 1.5 μΕ; flow rate: 0.2 mL/min; temperature: 30 °C; detection: 260 nm; analyte: amitriptyline loading range: 0.05 μg - 6.0 μg analyte on column; mobile phase: 0.05% TFA in 39% (for Commercially

Available 3 μπι Cis Column) or 37% (Product 4aa) acetonitrile. It is clear, as shown in Figure 4, that Product 4aa maintains nearly linear-isotherm behavior for amitriptyline at mass loads that approach those used in purification applications. EXAMPLE 43

BEH porous hybrid particles (20 g, Waters Corporation, Milford, MA; 4.0 μιη, 6.78 %C; SSA = 183 m ² /g; SPV = 0.70 cmVg; APD = 139 A) of the formula

(Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) was slurried in water (60 mL) for addition of 3-(trihydroxysilyl)propyl sulfuric acid (6 g, 50% solution). The solution was heated at 90 °C for 20 hours. The reaction was cooled and the product was filtered and washed with water and acetone. The product was then dried at 70 °C under a reduced pressure for 16 hours. The product had 7.29% C and an ion-exchange capacity of 0.160 mequiv/g by titration after subtracting the silanol contribution of a unbonded BEH particle. The surface coverage was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis to be 1.01 μπιοΐ/ιη ² .

EXAMPLE 44

Superficially porous silica particles (20 g, 1.3 μιη, SSA = 90-205 m ² /g; SPV = 0.1-

0.3 cm ³ /g; APD = 80-130 A) are reacted in a similar manner as detailed in Example 3 to yield a Cis bonded material that has an optimal concentration of an ionizable modifier, such as 4PE or APTES. This material (product 43a) is endcapped as detailed in Example 4, and evaluated as detailed in Examples 9-14, 41 and 42. The materials are evaluated as detailed in Examples 9-14, 41 and 42 and are compared to similar materials that do have the addition of the Component A ionizable modifier.

EXAMPLE 45

The process of Example 44 is performed using Superficially porous silica particles having a particle size of 0.3-2.0 μηι. The materials are evaluated as detailed in Examples 9- 14, 41 and 42. EXAMPLE 46

The process of Example 44 is performed using Superficially porous silica particles having a particle size of 2-3 μπι. The materials are evaluated as detailed in Examples 9-14, 41 and 42.

EXAMPLE 47

The process of Example 44 is performed using Superficially porous silica particles having a particle size greater than 3 μπι. The materials are evaluated as detailed in Examples 9-14, 41 and 42.

EXAMPLE 48

The process of Examples 44-47 are performed using a C4-C 12, C30, embedded polar, chiral, phenylalkyl, or pentafluorophenyl bonding and coatings in place of Cis bonding. The materials are evaluated as detailed in Examples 9-14, 41 and 42.

EXAMPLE 49

The process of Examples 44-48 are performed without the endcapping step prior to characterization. The materials are evaluated as detailed in Examples 9-14, 41 and 42. EXAMPLE 50

BEH porous hybrid particles (2.9 μπι, Waters Corporation, Milford, MA; 6.38%C; SSA = 86 m ² /g; SPV = 0.68 cm ³ /g; APD = 297 A) of the formula

(Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) were refluxed in toluene (9 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 2 hours. Upon cooling the Component A silane additive 2-(4- pyridylethyl)triethoxysilane was added and the reaction was heated to reflux for 1 hour. Upon cooling, imidazole (Aldrich, Milwaukee, WI) and the Component B silane tert- butyldimethylchlorosilane (TBDMCS, Gelest Inc., Morrisville, PA) or

octadecyltrichlorosilane (ODTCS, Gelest Inc., Morrisville, PA) was added. The reaction was then heated to reflux for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker) and then was hydrolyzed as detailed in Example 3, hydrolysis type C. The product was filtered and washed successively with toluene, 1 : 1 v/v/ acetone/water, and acetone. The product was dried at 70°C under reduced pressure for 16 hours. Reaction data are listed in Table 20. The surface coverage of these products was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis. Product 50b was further endcapped as detailed in Example 4 to yield a final carbon content of 10.52 %C.

Table 20

EXAMPLE 51

Superficially porous silica particles (1.35 um, SSA = 55 m ² /g; SPV = 0.15 cm ³ /g; APD = 107 A, 1.2 μπι non-porous core, 0.1 μπι thick porous shell) were refluxed in toluene (9 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 2 hours.

Upon cooling a Component A ionizable modifier 2-(4-pyridylethyl)triethoxysilane (4PE, Gelest Inc., Morrisville, PA) was added for product 51a and reaction was heated to reflux for 1 hour before cooling. No Component A ionizable modifier was added for product 51b. Imidazole (Aldrich, Milwaukee, WI) and octadecyltrichlorosilane (ODTCS, Gelest Inc., Morrisville, PA) were added. The reaction was then heated to reflux for 20 hours. The reaction was then cooled and the product was filtered and washed successively with water, toluene, 1 : 1 v/v acetone/water and acetone (all solvents from J.T. Baker) and then was hydrolyzed as detailed in Example 3, hydrolysis type C. The product was filtered and washed successively with toluene, 1 : 1 v/v/ acetone/water, and acetone. The product was dried at 70°C under reduced pressure for 16 hours. Reaction data are listed in Table 21. The surface coverage of these products was determined by the difference in particle %C before and after the surface modification as measured by elemental analysis. These products were further endcapped as detailed in Example 4.

Table 21

EXAMPLE 52

Following the protocol detailed in Example 13, peak capacity comparisons were made for Products 51a and 51b, as detailed in Table 22. The determination of peak capacity and the problems caused by poor peak shape and resulting poor peak capacities for basic analytes in low pH gradient separations is well known in the field of F1PLC and UPLC. Increased peak capacity ratios correlate with improved performance for basic analytes under these test conditions. Products 51a and 51b have the same feed material and were both similarly bonded, the only difference between these materials is the inclusion of the Component A ionizable modifier for product 51a. Improvements in peak capacity ratios were obtained for Product 51a over 51b, which is due to the introduction of the Component A ionizable modifier.

TABLE 22

EXAMPLE 53

Porous silica particles are hybrid coated, Cis-bonded and are endcapped in a process similar to the one detailed in US Patent 7,563, 367B to yield product 53a.

Alternatively, an ionizable modifier reagent, Component A (as detailed in Example 15) is added at different points in this process. Product 53b introduced the Component A additive before hybrid coating. Product 53c introduces the Component A additive before Ci8-bonding. Product 53d introduces the Component A additive before endcapping. Product 53e introduces the Component A additive after endcapping. The materials are evaluated as detailed in Examples 9-14, 41 and 42.

EXAMPLE 54

Superficially porous silica particles are hybrid coated, Cis-bonded and are endcapped in a process similar to the one detailed in US Patent 7,563, 367B to yield product 54a. Alternatively, an ionizable modifier, Component A (as detailed in Example 15) is added at different points in this process. Product 54b introduced the Component A additive before hybrid coating. Product 54c introduces the Component A additive before Ci8-bonding. Product 54d introduces the Component A additive before endcapping. Product 54e introduces the Component A additive after endcapping. The materials are evaluated as detailed in Examples 9-14, 41 and 42. EXAMPLE 55

BEH porous hybrid particles (4.0 μιη, 25 g, Waters Corporation, Milford, MA; 6.78%C; SSA = 183 m ² /g; SPV = 0.70 cm ³ /g; APD = 139 A) of the formula

(Oi.5SiCH2CH2SiOi.5)(Si02)4 (prepared following the method described in U.S. Patent 6,686,035) were refluxed in toluene (375 mL, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap. Upon cooling the zirconium n-propoxide (70% in n-propanol, 4.28 g, Gelest Inc., Morrisville, PA) was added and the reaction was stirred at ambient temperature for an hour and then heated to reflux overnight. The reaction was then cooled and the product was filtered and washed successively with toluene and 1% formic acid, and then was hydrolyzed in 1% formic acid for 1.5 hours at ambient temperature. Product 55a was filtered and washed with copious amounts of water and acetone. The product was dried at 80 °C under reduced pressure for 16 hours. EXAMPLE 56

Product 55a is further modified as detailed in Examples 1-8 and 15. The materials are evaluated as detailed in Examples 9-14, 41 and 42. EXAMPLE 57

The process of Examples 1, 3, 5, 7, 8, 15, 19, 21, 24, 27-29, 31-33, 35, 43-51, 53- 55 are performed by using one or more ionizable modifiers selected from the group (not limited to) alkoxides, halides, salts and complexes of zirconium, aluminum, cerium, iron, titanium, and other ionizable or amphoteric groups. These products are endcapped as detailed in Example 4. The materials are evaluated as detailed in Examples 9-14, 41 and 42. EXAMPLE 58

A chromatographic column containing a packed bed of 1-5 μιη chromatographic material that is Cis-bonded is evaluated as detailed in Examples 9-14, 41 and 42. This column is then flushed through with a dilute solution of a Component A, ionizable modifier in a suitable solvent for an extended time period to allow for incorporation of the ionizable modifier on the chromatographic bed. Examples of ionizable modifiers are included in Example 15 and 57. The column is further washed with a suitable solvent and is evaluated as detailed in Examples 9-14, 41 and 42. EXAMPLE 59

Ci8-bonded and endcapped 1-5 μπι chromatographic materials are modified with a Component A, ionizable modifier. Examples of chromatographic materials are included in Example 15. Examples of ionizable modifiers are included in Example 15 and 57. The materials are evaluated as detailed in Examples 9-14, 41 and 42 and are compared to the Ci8-bonded and endcapped material that does not contain an ionizable modifier.

EXAMPLE 60

The process of Example 58 and 59 are performed on superficially porous materials. Evaluations are performed as detailed in Examples 9-14, 41 and 42.

EXAMPLE 61

The process of Example 58-60 are preformed on chromatographic materials that are C4-C12, C30, embedded polar, chiral, phenylalkyl, or pentafluorophenyl bonding and coatings in place of Cis bonding. Evaluations are performed as detailed in Examples 9-14, 41 and 42.

EXAMPLE 62

Synthesis of DEAP HPCM Stationary Phases

DEAP HPCM stationary phases (i.e. Phase 1 A) were synthesized according to the following procedure: Step 1 : BEH porous particles (Waters Corporation, Milford, Mass,; 6.5% C; SSA = 75— 200 m ² /g ; SPV = 0.60— 0.75 cc/g; APD = 115— 310 A) of the formula

(0i.5SiCH2CH ₂ SiOi.5)(SiO2)4 (prepared following the method described in U.S. Pat. No. 6,686,035) were refluxed in toluene (5 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 1 hour. Upon cooling, redistilled (N,N-

Diethylaminopropyl)trimethoxysilane (DEAP, Silar Laboratories, Wilmington, NC) at 0.3 μπιοΐ/ιη ² was added and the reaction was heated to reflux for 2 hrs. The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from Fisher Scientific, Fairlawn, NJ). The product was then dried at 80° C under reduced pressure for 16 hrs.

Step 2: Material from Step 1 was refluxed in toluene (5 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 1 hour. Upon cooling, imidazole (Aldrich, Milwaukee, WI) and octadecyltrichlorosilane (Gelest Inc., Morrisville, PA) at 2.3 μπιοΐ/ιη ² were added and the reaction was heated to reflux for 16 hrs. The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from Fisher Scientific, Fairlawn, NJ). The material was then refluxed in acetone/aqueous 0.1 M ammonium bicarbonate (pH 10) solution for 20 hours at 50° C (hydrolysis). Following hydrolysis, the material was washed successively with 1 : 1 v/v acetone/water, and acetone (all solvents from Fisher Scientific, Fairlawn, NJ). The product was then dried at 80° C under reduced pressure for 16 hours.

Step 3 : Material from Step 2 was refluxed in toluene (5 mL/g, Fisher Scientific, Fairlawn, NJ) using a Dean-Stark trap for 1 hour. Upon cooling, imidazole (Aldrich, Milwaukee, WI) and triethylchlorosilane (TECS, Gelest Inc., Morrisville, PA) were added and the reaction was heated to reflux for 4 hrs. The reaction was then cooled and, imidazole and trimethylchlorosilane (Aldrich, Milwaukee, WI) were added to the reaction and the reaction was heated to reflux for an additional 16 hrs. The reaction was then cooled and the product was filtered and washed successively with toluene, 1 : 1 v/v acetone/water, and acetone (all solvents from Fisher Scientific, Fairlawn, NJ). The product was then dried at 80° C under reduced pressure for 16 hrs. Unless otherwise noted, all reagents described in the above procedure (Steps 1 through 3) were used as received. Those skilled in the art will recognize that equivalents exist, as such, although supplies and suppliers are listed, the listed supplies/suppliers should in no way be construed as limiting.

Stationary phases resulting from the above procedure were characterized in the following manner. The % C values were measured by Coulometric Carbon Analyzer (modules CM5300, CM5014, UIC Inc., Joliet, IL). The specific surface areas (SSA), specific pore volumes (SPV) 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 SPV was the single point value determined for P/Po > 0.98 and the APD was calculated from the desorption leg of the isotherm using the BJH method. Particle sizes were measured using a Beckman Coulter Multisizer 3 analyzer (30 μπι aperture, 70,000 counts; Miami, FL). The particle diameter (dp) was measured as the 50% cumulative diameter of the volume based particle size distribution. Total surface coverages of the octadecyltrichlorosilane were determined by the difference in particle %C before and after the surface modification as measured by elemental analysis. Those skilled in the art will recognize that equivalents of the following instruments exist and, as such, the instruments listed below are not to be construed as limiting. Information related to the DEAP HPCM phase, Phase 1 A, can be found below:

¹ As described in US7919177, US 7223473, US 6686035

Base Material DEAP Cl8

Example Particle Charge Coverage

(μπιοΐ/ιη ² ) (μπιοΐ/ιη ² )

1A Bl 0.3 2.4 EXAMPLE 63

Example Metabolites and Intermediates of the TCA Cycle

Standards were prepared in methanol and diluted with water to make a solution of 10 ng/mL . These analytes were then separated using a Waters ACQUIT Y UPLC I-Class LC system coupled with a Xevo TQ S tandem quadrupole mass spectrometer operated in ESI negative mode and in MRM acquisition mode. Details of the method are described below. Figure 5 presents MRM chromatograms of various TCA cycle metabolites and intermediates and the effectiveness of a mixed mode separation as performed with a DEAP HPCM column versus a Waters AC QUIT Y UPLC CSH CI 8 column of the same chromatographic particle size and column dimensions. Observed in Figure 5 is an increase in chromatographic retention of four molecules involved in the TCA cycle, as afforded by the DEAP HPCM column versus the commercially available ACQUITY UPLC CSH C18 column. The ACQUITY UPLC CSH CI 8 column is not effective in this mixed mode separation because it is packed with a charged surface reversed phase material that has an ionizable modifier with a pKa near 5. Accordingly, it provides little to no anionic retention under the conditions preferred for this negative ion mode LC-MS technique (Lauber, M. A.; Koza, S. M.; McCall, S. A.; Alden, B. A.; iraneta, P. C; Fountain, K. J., High- Resolution Peptide Mapping Separations with MS-Friendly Mobile Phases and Charge- Surface-Modified C18. Analytical chemistry 2013, 85 (14), 6936-44.; Gritti, F.; Guiochon, G., Adsorption behaviors of neutral and ionizable compounds on hybrid stationary phases in the absence (BEH-C18) and the presence (CSH-C18) of immobile surface charges. Journal of chromatography. A 2013, 1282, 58-71.)

LC Conditions

Column: DEAP HPCM 130A 1.65 μιη 2.1 χ 100 mm

Mobile Phase A: 100 % water titrated to pH 8.5 with ammonium hydroxide

Mobile Phase B: 40 % water 60% ACN 0.1 % ammonium hydroxide

Column Temperature: Injection Volume: 20 μΐ.

Sample Diluent: Water

Detection tandem quadrupole MS MRM mode ESI

negative mode

Gradient Table:

Time(min) Flow Rate(mL/min) %A %B Curve

Initial 0.450 100.0 0.00 Initial

1 5.00 0.450 65.0 35.0 6

1 7.00 0.450 5 .0 95 .0 6 1 8.00 0.450 100.0 0.0 6

25 .00 0.450 100.0 0.0 6

EXAMPLE 64

Example Nucleotides, Phosphorylated sugars and Other Biologically Relevant Acidic,

Polar Compounds

Standards were prepared in methanol and diluted with water to make a solution of 10 ng/mL . These analytes were then separated using a Waters ACQUIT Y UPLC I-Class LC system coupled with a Xevo TQS tandem quadrupole mass spectrometer operated in ESI negative mode and in MRM acquisition mode. Details of the method are described below. Figures 6 and 7 present MRM chromatograms of various acidic, polar,

biologically-relevant small molecules and a demonstration of the effectiveness of a mixed mode separation as performed with a DEAP HPCM column versus a Waters ACQUITY UPLC CSH C18 column of the same chromatographic particle size and column

dimensions. Observed in Figures 6 and 7 is the increase in chromatographic retention of two phosphorylated sugars, nucleotides and other biologically important molecules, as afforded by the DEAP HPCM column versus the commercially available ACQUITY UPLC CSH CI 8 column. LC Conditions

Column: DEAP HPCM 130A 1.65 μιη 2.1 χ 100 mm Mobile Phase A: 100 % water titrated to pH 8.5 with ammonium hydroxide Mobile Phase B: 40 % water 60% ACN 0.1 % ammonium hydroxide

Column Temperature: 45°C

Injection Volume: 20 μΐ.

Sample Diluent: Water

Detection: tandem quadrupole MS MRM mode ESI negative mode

Gradient Table:

Time(min) Flow Rate(mL/min) %B Curve Initial 0.450 0.00 Initial

1 5.00 0.450 65.0 35.0

1 7.00 0.450 5 .0 95 .0 1 8.00 0.450 100.0 0.0

25 .00 0.450 100.0 0.0

EXAMPLE 65

Glyphosate and Other Polar Pesticides

Standards were prepared in methanol and diluted with water to make a solution of

10 ng/mL. These analytes were then separated using a Waters ACQUITY UPLC I-Class LC system coupled with a Xevo TQ S tandem quadrupole mass spectrometer operated in ESI negative mode and in MRM acquisition mode. Details of the method are described below. Figure 8 presents this mixed mode separation for glyphosate and other polar pesticides. LC Conditions

Column: DEAP HPCM 130A 1.65 μιη 2.1 χ 100 mm Mobile Phase A: 100 % water Mobile Phase B: 40 % water 60% ACN 0.1 % ammonium hydroxide

Column Temperature: 45°C

Injection Volume: 20 μΐ,

Sample Diluent: Water

Detection: tandem quadrupole MS MRM mode ESI negative mode Gradient Table:

Time(min) Flow Rate(mL/min) %A %B Curve

Initial 0.450 100.0 0.00 Initial

1 5.00 0.450 65.0 35.0 6

1 7.00 0.450 5 .0 95 .0 6

1 8.00 0.450 100.0 0.0 6

25 .00 0.450 100.0 0.0 6

Incorporation By Reference

The entire contents of all patents published patent applications and other references cited herein are hereby expressly incorporated herein in their entireties by reference.

Equivalents

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.