High performance liquid chromatography (HPLC) separation combined with fluorescence detection and/or mass spectrometric detection is often applied in analysis of glycans in glycoproteins. The commonly used HPLC column chemistries are reversed-phase (RP), weak anion exchange (WAX), and hydrophilic interaction chromatography (HILIC). RP-HPLC is a common technology for the separation of glycans derivatized with a hydrophobic tag, which utilizes the non-covalent interaction between stationary phase and analyte to achieve the separation. HILIC and RP are complementary techniques in the separation of organic molecules with a broad band of polarity. HILIC-HPLC is the most widely used mothed for glycan analysis and is used to separate salivary and neutral glycans, providing advantages for rapid glycan profiling. HILIC facilitates the separation of glycans of the same charge according to their polarity and size. Glycans are eluted from a HILIC column in the order of increasing polarity. Under basic conditions, glycans are either partially or completely ionized and thus can be separated by anion-exchange chromatography. WAX-HPLC is an option for the separation of negatively charged glycans. WAX columns maintain their charge within a pH range. If the pH of the buffer used for a WAX column exceeds the capacity range of the matrix, the column will lose its charge distribution and molecules of interest may be lost.

Buffer solution is the key to control pH of the mobile phase to resist the change of pH value. Amerigo Scientific offers three buffer concentrates for HPLC analysis of glycans.

Buffers for HPLC analysis of Glycans

Product Name LudgerSep C Buffer x4 Concentrate LudgerSep N Buffer x40 Concentrate LudgerSep R BPT solvent x10 concentrate
Application Used in WAX-HPLC analysis of labeled glycans For preparation of LS-N buffer (50 mM ammonium formate buffer, pH 4.4) used in amide or HILIC-HPLC analysis of labeled glycans For preparation of butylamine/ orthophosphoric acid/ tetrahydrofuran solvent (BPT) used in monosaccharide HPLC analysis.
Description 2.0 M ammonium formate buffer/ solution pH 9.0 50 ml of x40 LS-N buffer (2.0 M ammonium formate buffer/solution) 50 mL of x10 LS-BPT solvent
Usage Dilute the whole contents of the bottle (50mL) with 150 mL HPLC grade water, then add 50 mL acetonitrile to make LS-C solvent (500 mM ammonium formate H 9, 20% acetonitrile v/v).
The 50 mL of x4 buffer will make 250 mL of LS-C solvent.
Dilute with de-gassed HPLC grade water (use 1 part of x40 buffer to 39 parts of water) to make LS-N buffer (50 mM ammonium formate, pH 4.4). The 50 ml of x40 buffer will make 2 liters of LS-N buffer. Dilute with de-gassed HPLC grade water use 1 bottle of LS-R-BPTX10 solvent to 450 mL of water.
The 50 mL of x10 solvent will make 500 mL of BPT solvent.
Storage Store unopened bottle below 25 °C. Store unopened bottle below 25 °C. Store unopened bottle at 4 °C.

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