The present disclosure generally relates to improved methods for separating and analyzing compounds by liquid chromatography, particularly when coupled with mass spectrometry. The methods include the addition of an additive to a mobile phase carrying a sample. The mobile phase additive may be effective for improving peak shapes of targeted analytes in acquired data, and/or eliminating or at least reducing ion suppression. The methods are particularly suited for anionic compounds such as phosphorylated compounds.

The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.

A packing material for liquid chromatography, which is excellent in durability, and a column for liquid chromatography, which is filled with the packing material, are provided. The packing material for liquid chromatography is characterized by comprising a hydrophilic resin containing a polyvinyl alcohol resin, to said hydrophilic resin an amino group represented by the formula (1) having been bonded through a spacer. In the formula (1), R.sup.1 represents a hydrogen atom or an alkyl group having 1 to 4 carbon atoms, R.sup.2 represents an alkyl group having 1 to 6 carbon atoms and having one or more hydroxyl groups, and represents a bonding position to the spacer.

A sol-gel sorbent or chromatography stationary phase is a particulate metal oxide gel containing polymeric segments uniformly distributed throughout the metal oxide gel. The metal oxide gel is an oxide from silicone or other metal oxide that can have one of the valence bonds attached to an organic group and the remainder occupied by oxygens that can be provided as an oxide or an alkoxide or aryl oxide of the polymeric segments. The particles are used for an SPE sorbent or as a packing for a reversed phase high-performance liquid chromatography (RP-HPLC), a normal phase high-performance liquid chromatography (NP-HPLC) column or a hydrophilic interaction liquid chromatography (HILIC) column.

The present disclosure relates to a method of separating a compound of interest, particularly unsaturated compound(s) of interest, from a mixture. The compound is separated using a column having a chromatographic stationary phase material for various different modes of chromatography containing a first substituent and a second substituent. The first substituent minimizes compound retention variation over time under chromatographic conditions. The second substituent chromatographically and selectively retains the compound by incorporating one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups, each group being optionally substituted with an aliphatic group.

In one aspect, the present invention provides a chromatographic stationary phase material for various different modes of chromatography represented by Formula 1: [X](W).sub.a(Q).sub.b(T).sub.c (Formula 1). X can be a high purity chromatographic core composition having a surface comprising a silica core material, metal oxide core material, an inorganic-organic hybrid material or a group of block copolymers thereof. W can be absent and/or can include hydrogen and/or can include a hydroxyl on the surface of X. Q can be a functional group that minimizes retention variation over time (drift) under chromatographic conditions utilizing low water concentrations. T can include one or more hydrophilic, polar, ionizable, and/or charged functional groups that chromatographically interact with the analyte. Additionally, b and c can be positive numbers, with the ratio 0.05?(b/c)?100, and a?0.

The invention relates to poly-amide bonded hydrophilic interaction chromatography (HILIC) stationary phases and novel HILIC methods for use in the characterization of large biological molecules modified with polar groups, known to those skilled in the art as glycans. The invention particularly provides novel, poly-amide bonded materials designed for efficient separation of large biomolecules, e.g. materials having a large percentage of larger pores (i.e. wide pores). Furthermore, the invention advantageously provides novel HILIC methods that can be used in combination with the stationary phase materials described herein to effectively separate protein and peptide glycoforms by eliminating previously unsolved problems, such as on-column aggregation of protein samples, low sensitivity of chromatographic detection of the glycan moieties, and low resolution of peaks due to restricted pore diffusion and long intra/inter-particle diffusion distances.

A method of purifying a desired protein from a preparation includes (a) providing the preparation in a form having less than about 5% of chromatin residing in an original production medium, (b) contacting the preparation with a nonionic organic polymer and a salt, a concentration of nonionic organic polymer being sufficient to precipitate the desired protein or cause its accretion on a hydrophilic surface, or maintain it in a precipitated state or accreted on the hydrophilic surface, the salt concentration being sufficient to produce greater than physiological conductivity, and (c) contacting the preparation with at least one electropositive surface, optionally in the presence of a salt concentration sufficient to produce greater than physiological conductivity, the desired protein does not substantially adsorb to the at least one electropositive surface while not preventing adsorption of acidic contaminants to the at least one electropositive surface.

A method of purifying a desired protein from a preparation includes (a) providing the preparation in a form having less than about 5% of chromatin residing in an original production medium, (b) contacting the preparation with a nonionic organic polymer and a salt, a concentration of nonionic organic polymer being sufficient to precipitate the desired protein or cause its accretion on a hydrophilic surface, or maintain it in a precipitated state or accreted on the hydrophilic surface, the salt concentration being sufficient to produce greater than physiological conductivity, and (c) contacting the preparation with at least one electropositive surface, optionally in the presence of a salt concentration sufficient to produce greater than physiological conductivity, the desired protein does not substantially adsorb to the at least one electropositive surface while not preventing adsorption of acidic contaminants to the at least one electropositive surface.

The present disclosure relates to a method of separating a compound of interest, particularly unsaturated compound(s) of interest, from a mixture. The compound is separated using a column having a chromatographic stationary phase material for various different modes of chromatography containing a first substituent and a second substituent. The first substituent minimizes compound retention variation over time under chromatographic conditions. The second substituent chromatographically and selectively retains the compound by incorporating one or more aromatic, polyaromatic, heterocyclic aromatic, or polyheterocyclic aromatic hydrocarbon groups, each group being optionally substituted with an aliphatic group.