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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