JPS6125059A - Method and apparatus for analyzing saccharide - Google Patents

Abstract

PURPOSE:To perform an analysis handily at a low cost with a high accuracy and a high reproducibility, by eluting a sample for a liquid chromatography using a mobile phase containing a reaction reagent to cool and measure the elution liquid by an excited light and/or ultraviolet rays after a heating reaction thereof. CONSTITUTION:A measuring section 4 is connected to a liquid chromatography 1, a reaction section 2 and a cooling section 3. Then, the temperatures of a column 9, a reaction tube 10 and a cooling tube 20 are kept at a specified level and a mobile phase 6 containing amino acid and boronic acid of a reaction reagent is discharged from a reaction liquid discharge tube 19 with a pump 7 via the column 9, the reaction tube 10, the cooling tube 20 and a photometer 17 to establish a steady state. Then, a sample is injected from a sample injection section 8 to be separated while sugars pass through the column 9 and the sugars are subjected to a heating reaction with a reaction reagent in the reaction tube 10 to be converted into a violet absorbing and fluorescent substance whose concentration is detected with a fluorescence photometer and/or a ultrasonic absorption photometer 17. This eliminates the passage for supply of the reaction agent to enable a highly accurate analysis free of noise and drift while ensuring a very high reproducibility.

Description

[Detailed description of the invention] (b) Industrial application fields The present invention relates to a method and apparatus for analyzing sugars.

(b) Prior Art The inventor of the present invention has already disclosed a method and apparatus for analyzing sugars in Japanese Patent Application Laid-Open No. 58-216953.

That is, a reaction reagent consisting of a boric acid aqueous solution containing a basic amino acid was added to the column eluate obtained by subjecting Sample 1 containing sugars to liquid chromatography, and after a heating reaction was performed, it was cooled and excitation light was applied. A method of analyzing saccharides by measuring the fluorescence intensity and/or measuring the absorbance by applying ultraviolet rays, and a liquid chromatography device itself.
A reaction solution flow path is extended from the column eluent flow path, a supply flow path for a boric acid aqueous solution containing a basic amino acid is connected to this flow path, and then a heating section, a cooling section, a fluorescence measurement section, and/or This is a saccharide analyzer equipped with an ultraviolet absorption photometry section in this order.

In the above analysis technique, the reaction reagent consisting of a boric acid aqueous solution containing a basic amino acid is added to the eluent obtained by subjecting the sugar-containing sample to liquid chromatography and reacted. It is necessary to provide a supply flow path for adding the reaction reagent to the solution 1IIE, and noise and drift may occur in the obtained chromatogram due to insufficient mixing of the reaction reagent and the solution 1IIE.

(c) Purpose of the Invention The purpose of the present invention is to improve the above-mentioned prior art analytical method and device, and to further simplify the prior art analytical method and its device, and to achieve analysis with high precision and good reproducibility. An object of the present invention is to provide a method for analyzing saccharides and an apparatus therefor.

(d) Structure of the Invention This invention involves eluating a sample containing sugars through liquid chromatography using a mobile phase containing reaction reagents, amino acids and boric acid, and heating the resulting eluate to react. After the reaction between the reagent and the saccharide is performed, the reaction solution is cooled, and the resulting reaction solution is exposed to excitation light and the fluorescence intensity is measured, and/or the saccharide is analyzed by irradiating it with ultraviolet light and measuring the absorbance. A saccharide analysis method and analytical equipment directly used to carry out this analysis method, including a mobile phase container containing amino acids and boric acid as reaction reagents, a mobile phase liquid pump, a sample injection unit, and a separation column. The present invention provides a saccharide analysis device in which a liquid chromatography device, a reaction section equipped with a heating tank, a cooling section, and a fluorescence measurement section and/or an ultraviolet absorption measurement section are connected in this order.

The inventor of this invention arrived at the analytical method of this invention by discovering that even if a reaction reagent is mixed in the mobile phase, there is no adverse effect on the separation of sugars in a separation column. Since the reaction reagent is mixed in the mobile phase, there is no noise or drip in the chromatogram caused by the mixing of the eluent and reaction reagent as in the prior art, and there is no flow path for supplying the reaction reagent. It has the advantage that it is not necessary to provide

The amino acids used in this invention include arginine,
Examples include lysine and histidine.

As a mobile phase, amino acids with a concentration of 0.01 to 5% and 0.
．． An aqueous solution containing 0.05 to 0.5M boric acid whose pH is adjusted to about 7 to 10 with an alkali such as potassium hydroxide is used.

For saccharide-containing samples, use the above mobile phase and Shim −+1a
It is eluted with a commonly used separation column such as ck ISA-07/S 2504 (manufactured by Shimadzu). etc. This elution is carried out at temperatures between room temperature and 70°C. The resulting eluate is then heated to cause the saccharide to react with the reaction reagent. This reaction is preferably carried out at 140-180°C for 3-5 minutes. The generated reaction solution is cooled to room temperature and introduced into a fluorometer and/or ultraviolet absorption photometer to measure fluorescence and/or absorption, thereby qualitatively and/or quantitatively analyzing saccharides.

On the other hand, the main structural feature of the analyzer of the present invention is that the reaction reagent is contained in the mobile phase as described above, and therefore, compared to the apparatus of the prior art, the reaction reagent supply channel is separately connected. There is no need to provide one, making it extremely simple. And since this invention is continuous and transient,
Fast analysis is possible and can be fully automated.

Examples of sugars that can be analyzed using the analytical method and apparatus of the present invention include monosaccharides such as glucose, mannose, galactose, fructose, and rhamnose, maltose,
Oligopolysaccharides such as lactose and maltotriose, amino sugars such as glucosamine and galactosamine, glucuronic acid,
Examples include uronic acids such as galacturonic acid, and can be applied to all sugars. It is particularly suitable for analyzing biological samples that contain trace amounts and/or multiple components of these saccharides.

(e) Examples This invention will be explained below based on examples shown in the figures.
This invention is not limited by this.

FIG. 1 shows an explanatory diagram of the configuration of an embodiment of the analyzer of the present invention. This analyzer includes a liquid chromatograph (1),
Reaction 811 (2), cooling section (3) equipped with cooling pipe (a)
) and a fluorescence measurement section and/or an ultraviolet absorption measurement section (4) are connected in this order through a flow path.

The liquid chromatograph (1) is equipped with a mobile phase container (6) containing a mobile phase (5) containing reaction reagents amino acids and boric acid, a mobile phase liquid pump (7), a sample injection part (8), and a constant temperature bath. It consists of a separation column (9).

The reaction section (2) includes a reaction tube aO), an electric heater as a heat source (
It is composed of an inner tank 04) in which the heat sensitive body 11) and a temperature control heat sensitive body (I2) are housed together with a heat medium 03), and an outer tank (16) in which this is housed via a heat insulating material 05).

The measuring section (4) is composed of a fluorometer and/or an ultraviolet absorption photometer (171 and a recorder 08 attached thereto) and a reaction liquid discharge pipe Oa.

Next, a method for analyzing saccharides using the above-mentioned apparatus will be explained.

First, while maintaining the temperature of the separation column (9), reaction tube 00) and cooling tube at a predetermined temperature, the mobile phase (6) is pumped (by force) into the separation column (9), reaction tube (1O+ ,
The reaction liquid is discharged from the reaction liquid discharge tube U via the cooling tube (A) and the photometer (+71) to maintain a steady state.Then, the sugar-containing sample is injected from the sample injection part (8), and the sugars in the sample are transferred to the separation column. (9), and then the reaction tube 0
For example, the saccharides separated in 0) and the reaction reagent are
Reacting for 5 minutes at 0° C. converts the sugars into UV-absorbing and fluorescent substances, and the concentration is detected on a fluorometer and/or UV absorption photometer.

Next, an example of analysis of saccharides using the above device will be shown.

Analysis conditions Liquid chromatograph device: Shimadzu high performance liquid chromatograph LC-3A Separation column: Shim-pack I 5A-0
7/82504 Mobile phase: 0,35M boric acid and 0,1% L
-Arginine-containing aqueous solution (pH=8. with potassium hydroxide)
5) Mobile phase flow rate: 1. Otf/min Separation column temperature: 265°C Reaction tube temperature: 150°C Detector: Fluorometer (island) ↑RF-530. Excitation wavelength 3
20 nm, fluorescence wavelength 430 nm) Sample: Aqueous solution of a mixture of maltose, mannose, galactose, xylose and glucose (the amount of each sugar is lnmol) The chromatogram obtained as a result of the above analysis is shown in FIG. As is clear from Figure 2, analysis was possible with high accuracy without noise or drift. When this analysis was repeated, the reproducibility was extremely good.

(F) Effects of the Invention According to the present invention, saccharides can be analyzed more easily and at lower cost with higher accuracy and higher reproducibility than the conventional analytical techniques described above.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the configuration of an embodiment of the analyzer of the present invention;
FIG. 2 is a chromatogram obtained by an analytical example of the present invention. (1)・・・Liquid chromatography, (2)・・・
...Reaction section, (3)...Cooling section, (4)...
...Fluorescence measurement section and/or ultraviolet absorbance measurement section,
(5)... Mobile phase containing reaction reagent, (6)...
...mobile phase container, (power ... mobile phase liquid delivery pump, (8) ...sample injection part, (9) ...
Separation column with constant temperature bath, (g))...Reaction tube, (
11)... Electric heater, (14)...
・Inner tank, 06)... Outer tank, Q71...
Fluorometer and/or UV absorption photometer, (18)...
... Recorder, ■ ... Reaction liquid discharge pipe, (a) ... Cooling pipe.

Claims (1)

[Claims] 1. A saccharide-containing sample is eluted by liquid chromatography using a mobile phase containing reaction reagents, amino acids and boric acid, and the resulting eluate is heated to elute the reaction reagent. A process for analyzing saccharides, which consists of performing a reaction with saccharides, cooling them, and then applying excitation light to the resulting reaction solution and measuring the fluorescence intensity, and/or applying ultraviolet rays and measuring absorbance to analyze the saccharides. Analysis method. 2. A liquid chromatograph device consisting of a mobile phase container containing amino acids and boric acid as reaction reagents, a mobile phase pump, a sample injection section, and a separation column, a reaction section equipped with a heating tank, a cooling section, and a fluorescence A saccharide analysis device in which a photometric unit and/or an ultraviolet absorption photometric unit are connected through this route.