HPLC and UHPLC Columns for Glycan Analysis

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Most glycoproteins carry a heterogeneous mixture of oligosaccharides, and even the individual glycosylation sites of pure glycoproteins are often heterogeneously glycosylated. The structural diversity of oligosaccharides is caused by linkage variation, difference in size and charge number, and difference in monosaccharide composition of glycans. Many analytical technologies have been employed in the structural characterization of glycans, such as nuclear magnetic resonance (NMR), mass spectrometry (MS), and gas chromatography-mass spectrometry (GC-MS).

Depending on its nature, the glycan moiety can be released from the protein by enzymatic cleavage or chemical methods. Free glycans released from glycoproteins are usually found in solutions containing salts, detergents, proteins, peptides, amino acids, etc. These contaminants must be removed prior to further glycan analysis. A crucial part of glycan analysis is the separation of the glycan mixture into homogeneous glycan fractions. The most widely used methods for glycan separation are weak anion exchange (WAX), gel filtration, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and normal-phase high-performance liquid chromatography (NP-HPLC). The selection of appropriate methods should be based on the specific anlysis requirements and properties of the glycans to be analyzed.

Fluorescent labeling of the glycan moiety usually improves the detection and the most commonly used labels are 2-aminobenzoic acid (2-AA) and 2-aminobenzamide (2-AB). HPLC-based methods are usually applied with fluorescent-labeled oligosaccharides for the separation of sialylated and neutral glycans from various biological samples such as recombinant therapeutic proteins and growth hormones.

HPLC Columns for Glycan Analysis

Amerigo Scientific offers high quality HPLC columns for the separation of negatively charged glycans, fluorophore labelled glycans, 1,2-diamino-4,5-methylenedioxybenzene (DMB) labelled sialic acids, or monosaccharides.

Column	C2 anion exchange HPLC column	C3 anion exchange HPLC column	N1 amide HPLC column	N2 amide HPLC column	R1 HPLC column	R2 HPLC column
Application	Separation of negatively charged glycans into neutral, mono, di, tri and tetra species		Separation of fluorophore labelled glycans according to size and shape		Separation of DMB labelled sialic acids	Monosaccharide analysis
Description	It contains polystyrene particles with a macroporous polymeric anion exchange coating optimized for anion exchange chromatography.	It contains macroporous anion exchange particles optimized for anion exchange chromatography of complex glycan mixtures.	They contain 5 um particles with a polymeric amide coating suitable for low resolution chromatography of complex glycan mixtures where UHPLC is not available.	They contain 3 um particles with a polymeric amide coating suitable for low resolution chromatography of complex glycan mixtures where UHPLC is not available and quicker gradients than N1 are required.	It contains particles with an octadecylsilane coating optimized for hydrophobic chromatography.	It contains particles with an octadecylsilane coating optimized for hydrophobic chromatography. It is optimized to handle efficient elution of the monosaccharides from the free dye peak.
Pore size	1000 angstroms	1000 angstroms	80 angstroms	80 angstroms	120 angstroms	175 angstroms
Particle size	8 μm	10 μm	5 μm	3 μm	3 μm	3 μm
Column Dimensions	4.6 mm x 50mm; 4.6 mm x 150 mm	7.5 mm x 75 mm	4.6 mm x 10 mm; 4.6 mm x 250 mm	4.6 mm x 150 mm; 2.1 mm x 150 mm	4.6 mm x 150 mm	4.6 mm x 150 mm
Flow Rates	1 mL/min	0.3–1.2 mL/min	0.4 mL/min typical	0.4 mL/min typical	0.3 mL/min	0.3-2 mL/min
Pressure	Max 3000 psi (207 bar)	Max 2175 psi (150 bar)	Max 2175 psi (150 bar)	Max 2900 psi (200 bar)	Max 5800 psi (400 bar)	Max 5800 psi (400 bar)
pH Range	1-14	2-12	2-7.5	2-7.5	2-8	1-11
Temperature	10-80 °C	10-45 °C	30 °C typical (range 10-80 °C)	30 °C typical (range 10-50 °C)	60 °C max	60 °C max
Solvents	Acetonitrile 20-500 mM ammonium formate or ammonium acetate solvents	Acetonitrile 20-500 mM ammonium formate or ammonium acetate solvents	Acetonitrile 50-250 mM ammonium formate pH4.4	Acetonitrile 50-250 mM ammonium formate pH4.4	Acetonitrile + methanol mix	Solvent A: 0.2% butylamine / 0.5% phosphoric acid / 1% tetrahydrofuran in purified water Solvent B: Acetonitrile
Companion Products	LudgerSep C Buffer x4 Concentrate		LudgerSep N Buffer x40 Concentrate		--	LudgerSep R BPT solvent x10 concentrate

UHPLC Columns for Glycan Analysis

Compared with HPLC, ultrahigh performance liquid chromatography (UHPLC) uses smaller diameter packing materials and higher pressures. In UHPLC, the average particle size is usually 1.7 μm in diameter, which is smaller than the 3–5 μm particle diameter of HPLC column. The main advantage of UHPLC over HPLC is speed. Additionally, complex mixtures can be resolved better using UHPLC, and solvent consumption is less in UHPLC than in HPLC.

Amerigo Scientific offers UHPLC columns for the separation of the seven main monosaccharides found in most N-linked and O-linked glycans or the separation of DMB labelled sialic acids.

Column	uR2 UHPLC column for monosaccharide analysis	uR2 DMB UHPLC column for sialic acid analysis
Application	Monosaccharide analysis	Separation of DMB labelled sialic acids
Description	It contains particles with an octadecylsilane coating optimized for hydrophobic chromatography. It is optimized to handle efficient elution of the monosaccharides from the free dye peak.	It contains particles with an octadecylsilane coating optimized for hydrophobic chromatography.
Pore size	175 angstroms	176 angstroms
Particle size	1.9 μm	1.9 μm
Column Dimensions	2.1 mm x 50 mm	2.1 mm x 100 mm
Flow Rates	0.4 mL/min	0.25 mL/min
Pressure	Max 18000 psi (1250 bar)	Max 18000 psi (1250 bar)
pH Range	1-11	1-11
Temperature	60 °C max	60 °C max
Solvents	Solvent A: 0.2% butylamine / 0.5% phosphoric acid / 1% tetrahydrofuran in purified water Solvent B: Acetonitrile	Acetonitrile + methanol mix
Companion Products	LudgerSep R BPT solvent x10 concentrate	--

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