Method for analysis of saccharides

A method for separation analysis of reducing sugars contained in a sample by column chromatography, comprising

• loading the sample on an anion exchanger resin column, and washing the column by allowing a sufficient volume of a first mobile phase to flow consisting of an aqueous solution of a predetermined concentration of boric acid comprising at least a predetermined concentration of a water-soluble inorganic salt through the column;
• supplying to the column continuously at a predetermined flow rate a second mobile phase comprising the salt at an elevated concentration compared with the first mobile phase to eluate reducing sugars;
• leading the eluate from the column continuously to a flow path;
• continuously adding at least one basic amino acid at a predetermined rate to the eluate at a predetermined position of the flow path to make a mixture solution which continuously flows through the flow path;
• heating the mixture solution by letting the mixture solution flow through a heating zone at a predetermined temperature of the flow path over a predetermined length of time;
• and
• continuously measuring and recording the intensity of fluorescent light emitted from the mixture solution while continuously irradiating the mixture solution thus heated and flowing through the flow path with excitation light at a predetermined position of the flow path;
• wherein the pH of the first and the second mobile phases is 7.5-9.5.

A method for separation analysis of reducing sugars contained in a sample by column chromatography, comprising

• loading the sample on an anion exchanger resin column, and washing the column by allowing a sufficient volume of a first mobile phase to flow consisting of an aqueous solution of a predetermined concentration of boric acid containing at least a predetermined concentration of a water-soluble inorganic salt through the column;
• supplying to the column continuously at a predetermined flow rate a second mobile phase containing the salt at an elevated concentration compared with the first mobile phase to eluate reducing sugars;
• leading the eluate from the column continuously to a flow path;
• continuously adding at least one basic amino acid at a predetermined rate to the eluate at a predetermined position of the flow path to make a mixture solution which continuously flows through the flow path;
• heating the mixture solution by letting the mixture solution flow through a heating zone at a predetermined temperature of the flow path over a predetermined length of time;
• and
• continuously measuring and recording the intensity of fluorescent light emitted from the mixture solution while continuously irradiating the mixture solution thus heated and flowing through the flow path with excitation light at a predetermined position of the flow path,
• wherein the water-soluble inorganic salt is sodium chloride.

A method for separation analysis of reducing sugars contained in a sample by column chromatography, comprising

• loading the sample on an anion exchanger resin column, and washing the column by allowing a sufficient volume of a first mobile phase to flow consisting of an aqueous solution of a predetermined concentration of boric acid containing at least a predetermined concentration of a water-soluble inorganic salt through the column;
• supplying to the column continuously at a predetermined flow rate a second mobile phase containing the salt at an elevated concentration compared with the first mobile phase to eluate reducing sugars;
• leading the eluate from the column continuously to a flow path;
• continuously adding at least one basic amino acid at a predetermined rate to the eluate at a predetermined position of the flow path to make a mixture solution which continuously flows through the flow path;
• heating the mixture solution by letting the mixture solution flow through a heating zone at a predetermined temperature of the flow path over a predetermined length of time;
• and
• continuously measuring and recording the intensity of fluorescent light emitted from the mixture solution while continuously irradiating the mixture solution thus heated and flowing through the flow path with excitation light at a predetermined position of the flow path;
• wherein the reducing sugars include mannose-6-phosphate.

An aqueous solution for use as the first mobile phase in a separation analysis of reducing sugars contained in a sample by column chromatography, comprising

• loading the sample on an anion exchanger resin column, and washing the column by allowing a sufficient volume of a first mobile phase to flow consisting of an aqueous solution of a predetermined concentration of boric acid containing at least a predetermined concentration of a water-soluble inorganic salt through the column;
• supplying to the column continuously at a predetermined flow rate a second mobile phase containing the salt at an elevated concentration compared with the first mobile phase to eluate reducing sugars;
• leading the eluate from the column continuously to a flow path;
• continuously adding at least one basic amino acid at a predetermined rate to the eluate at a predetermined position of the flow path to make a mixture solution which continuously flows through the flow path;
• heating the mixture solution by letting the mixture solution flow through a heating zone at a predetermined temperature of the flow path over a predetermined length of time;
• and
• continuously measuring and recording the intensity of fluorescent light emitted from the mixture solution while continuously irradiating the mixture solution thus heated and flowing through the flow path with excitation light at a predetermined position of the flow path;
• and wherein the aqueous solution for use as the first mobile phase contains 50-150 mM boric acid and 10-30 mM sodium chloride.

An aqueous solution for use to prepare the second mobile phase by mixing with the first mobile phase in a method for separation analysis of reducing sugars contained in a sample by column chromatography, comprising

• loading the sample on an anion exchanger resin column, and washing the column by allowing a sufficient volume of a first mobile phase to flow consisting of an aqueous solution of a predetermined concentration of boric acid comprising at least a predetermined concentration of a water-soluble inorganic salt through the column;
• supplying to the column continuously at a predetermined flow rate a second mobile phase comprising the salt at an elevated concentration compared with the first mobile phase to eluate reducing sugars;
• leading the eluate from the column continuously to a flow path;
• continuously adding at least one basic amino acid at a predetermined rate to the eluate at a predetermined position of the flow path to make a mixture solution which continuously flows through the flow path;
• heating the mixture solution by letting the mixture solution flow through a heating zone at a predetermined temperature of the flow path over a predetermined length of time;
• and
• continuously measuring and recording the intensity of fluorescent light emitted from the mixture solution while continuously irradiating the mixture solution thus heated and flowing through the flow path with excitation light at a predetermined position of the flow path;
• and wherein the aqueous solution for use to prepare the second mobile phase contains 50-150 mM boric acid and 200-250 mM sodium chloride.