JPS6367662B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION This invention relates to a method for analyzing sugars. More specifically, this invention subjects a sample containing sugars to liquid chromatography, adds a reaction reagent of a boric acid aqueous solution containing monoethanolamine or a boric acid buffer solution containing taurine to the column eluent, and heats the sample. The present invention relates to an ultraviolet absorption analysis method for saccharides, which is characterized by qualitatively or quantitatively analyzing saccharides by cooling after a reaction, applying ultraviolet rays, and measuring the absorbance.

Conventionally, sugars have been detected using a liquid chromatograph using a differential refractometer. However, since this differential refractometer cannot employ the gradient elution method, it is not easy to analyze samples containing multi-component sugars such as biological samples. In other words, in liquid chromatography, even if sugars are eluted by continuously changing the solvent composition, there is usually a large difference in the refractive index of each solvent. Analysis of sugars was almost impossible due to the large difference. Furthermore, differential refractometers have inherently low sensitivity, making it difficult to detect trace amounts of sugars.

In addition, the invention of a method and apparatus for reacting sugars with a specific reaction reagent to generate fluorescence in sugars that do not themselves emit fluorescence, and detecting the fluorescence intensity to analyze sugars is known (Japanese Patent Laid-Open No. No. 55-70739). Here, the inventors of this invention conducted further research and discovered that saccharides that do not substantially absorb ultraviolet rays by themselves strongly absorb ultraviolet rays by reacting saccharides with a specific reaction reagent, and specifically analyzed the saccharides. This provides a method that can be used in a variety of ways.

According to this invention, sugars can be analyzed with higher sensitivity and better linearity than in the case of fluorescence photometry, and furthermore, a conventionally widely used ultraviolet absorption spectrophotometer can be used. It has the advantage of

One of the main structural features of the analyzer for carrying out the method of this invention is that the mixture of column eluent and reaction reagent moves through a reaction liquid flow path to a heating tank and then to a cooling tank. This makes it possible to carry out reactions and perform ultraviolet absorption spectrophotometric analysis, and also allows the use of a gradient elution method in liquid chromatography, which enables the analysis of multi-component saccharides such as those found in living organisms. can be analyzed with high precision all at once.

Another structural feature of the analyzer for carrying out the method of this invention is that the flow of the sample liquid is continuous and one-time, from the supply of the column eluent and reaction reagent to the discharge of the reaction liquid. This enables high-speed analysis and is suitable for complete automation.

Examples of sugars that can be analyzed using the analytical method of this invention include monosaccharides such as glucose, mannose, galactose, fructose, rhamnose, glucosamine, and glucuronic acid; oligopolysaccharides such as maltose, lactose, and maltotriose; It can be applied to sugar. It is particularly suitable for analyzing biological samples that contain multiple components of these saccharides.

As the reagent for reacting with saccharides used in this invention, for example, a buffer solution containing an alkylamine derivative such as monoethanolamine or taurine is used. Among these, the use of monoethanolamine as the alkylamine derivative dissolved in a boric acid solution gives the most favorable results. In this case, analysis is possible if the concentrations of monoethanolamine and boric acid in the reagent are each in the range of 0.5 to 5.0%. Further, the mixing ratio of the reaction reagent to the column eluent is preferably in the range of 50 to 75%. The reaction is preferably carried out at 140°C to 180°C for 3 to 5 minutes. After the reaction, the mixture is cooled to room temperature and introduced into an ultraviolet absorption photometer.

The present invention will be described in detail below based on embodiments shown in the figures. Note that this invention is not limited by this.

In FIG. 1, an automatic saccharide analyzer 1 includes a high-performance liquid chromatograph main body 25 and a reaction liquid pipe 9 extending to an eluent pipe 3 of a column 2.
, a reaction reagent supply pipe 4 which is connected to the reaction liquid pipe, and a heating tank 6, a cooling tank 7, through which the reaction liquid pipe 9 passes in order after the joint connection part (mixing part) 5. It mainly consists of an ultraviolet absorption photometer 8 and a reaction liquid discharge pipe 10 further extending from a reaction liquid pipe 9.

The reaction reagent supply line 4 is provided with a pump 11 and a damper 12, and supplies an aqueous boric acid solution containing monoethanolamine as a reaction reagent to the reaction liquid line 9, where it is mixed with the column eluent. Normally, a three-way conduit is used for the merging connection part 5 of both flow paths.

The heating tank 6 is filled with tin, which has good heat transfer, as a heating medium, and an electric heater 17 and a reaction liquid pipe 9 as a heat source are inserted into this heating medium. The insertion portion 13 of this reaction liquid conduit is formed into a coil shape with a predetermined length. In addition, 18 is a heat sensitive body for temperature control, 19 is an inner tank part, 20 is an outer tank part, and 21 is a heat insulating material filled between both tanks.

The cooling tank 7 introduces circulating cooling water, and the reaction liquid pipe 9 is inserted into this cooling water. The insertion portion 14 of this reaction liquid conduit is also formed into a coil shape with a predetermined length.

The ultraviolet absorption photometer 8 is equipped with a recorder 16 and the like, and a resistance tube 2 is connected to the reaction liquid discharge pipe 10.
2 is interposed.

Next, automatic saccharide analyzer 1 consisting of the above configuration
We will explain how to analyze sugars using

First, the pump 11 of the reaction reagent supply line 4 and the pump 23 of the high performance liquid chromatography apparatus main body 25 are operated in advance to supply a reaction reagent consisting of a boric acid aqueous solution containing monoethanolamine and a column eluent to the reaction liquid pipe. 9 and maintains a steady state. Next, the sample is introduced into the column 2 from the main body injection part 24.
When injected into the column, the sample is separated into components by column,
It is eluted from the column and enters the reaction liquid line 9 through the eluent line 3, where it is mixed with a reaction reagent and heated to 150°C for about 5 minutes in a heating tank 6 to react.
Furthermore, by passing through a cooling tank 7, it is cooled to room temperature. Through this passage, the sample component (sugar) is converted into an ultraviolet absorbing substance, and the concentration is detected by an ultraviolet absorption photometer 8.

In this way, sugar analysis becomes possible with high sensitivity. Furthermore, after mixing the column eluent with the reaction reagent,
Automatically reacts while being moved and discharged in a transitory manner,
Cooling and ultraviolet absorption photometry are performed, so
Suitable for full automation. Of course, there is no point where the movement is interrupted, so analysis can be performed at high speed.

More importantly, since a gradient elution method can be employed in a liquid chromatograph, multi-component saccharides such as saccharides in living organisms can be analyzed at once with high precision.

Experimental Example 1 Using the automatic analyzer 1 shown in FIG. 1, the following analysis results were obtained.

Shimadzu high performance liquid chromatograph LC-3A [Analysis column 2 is Shimadzu LC column ISA-07/S2504] was used, and the mobile phases were A: 0.2M boric acid aqueous solution (adjusted to pH 8.0 with potassium hydroxide) and B. :0.45M boric acid aqueous solution (adjusted to PH=9.0 with potassium hydroxide)
Gradient program: A/B, 100/0
It was used at 1%/min---→40/6010%/min---→0/100. The flow rate was 0.6 ml/min through the column. However, the column temperature was 60°C.

The samples are A: cellobiose, B: maltose, C: lactose, D: rhamnose, E: ribose, F: mannose, G: fructose, H:
An aqueous solution containing 0.5 mg/ml of each of galactose, I: xylose, and J: glucose was injected into a 30μ column.

Further, a 5.0% monoethanolamine-3.0% boric acid aqueous solution was used as a reaction reagent at a flow rate of 0.6 ml/min, and the reaction temperature was 150°C. The reaction pipe section 13 used had an inner diameter of 0.8 mm and a length of 10 m.

A Shimadzu UVD-1 model was used as the ultraviolet absorption photometer, and for comparison, the ultraviolet absorption photometer 8 was used.
A Shimadzu FLD-1 type fluorometer was inserted between the tube and the cooling tank 7, and the fluorescence intensity was measured at the same time [excitation light:
300-400nl (maximum 360nm), fluorescence measurement: EM-
Uses 4 filters (transmits approximately 430 nm or more). The sensitivity range for both was the same X8, and charts of the measurement results are shown in Figure 2 (fluorescence intensity) and Figure 3 (ultraviolet absorption intensity).

The results showed that although the peak width was wider because the UV absorbance photometer was connected after the fluorometer, the peak height of each component was higher in the UV absorbance, indicating higher sensitivity. It can be seen that particularly D: rhamnose, G: fructose, H: galactose, and I: xylose have high sensitivity.

Experimental Example 2 As in Experimental Example 1, a fluorometer and an ultraviolet absorption photometer were connected in series, and three saccharide mixed aqueous solutions were analyzed under the following conditions.

Analysis conditions Equipment: Shimadzu high performance liquid chromatograph LC
-3A Analytical column: Shimadzu LC column PNH ₂ -10/S2504 Mobile phase: Acetonitrile 70/Water 30 Mobile phase flow rate: 0.6ml/min Column temperature: Room temperature Fluorometer and UV spectrophotometer: Shimadzu FLD
-1 and UVD-1 However, the sensitivity ranges of both photometers are X2 and X4, respectively.

Sample: K: Glucose, L: Maltose, M: Maltotriose Sample concentration: Approximately 1 μmol/ml each Sample injection amount: 10 μ Reaction conditions Reaction reagent: 5.0% monoethanolamine
3.0% boric acid aqueous solution Reaction temperature: 150℃ Reaction tube: inner diameter 0.8 mm, length 10 m Reaction reagent flow rate: 0.6 ml/min Measurement results of fluorescence and ultraviolet absorbance were measured in the fourth column.
It is shown in FIG. In this case as well, ultraviolet absorption photometry clearly has higher sensitivity than fluorescence photometry, and the difference is larger than in Example 1.

Experimental Example 3 As in Example 1, a fluorometer and an ultraviolet absorption photometer were connected in series, and an analysis was performed under the following conditions using glucose aqueous solutions (content 0.4 μg to 7 μg) with varying concentrations as samples. Ivy.

Analysis conditions Equipment: Shimadzu high performance liquid chromatograph LC
-3A Analytical column: Shimadzu LC column ISA-07/S2504 Mobile phase: 0.5M-boric acid (adjusted to PH = 9.5 with potassium hydroxide) Mobile phase flow rate: 0.5ml/min Column temperature: 60℃ Fluorometer and UV absorption photometer: Shimadzu FLD
-1 and UVD-1 Reaction conditions Reaction reagent: 5.0% monoethanolamine -
3.0% boric acid aqueous solution Reaction temperature: 150℃ Reaction tube: inner diameter 0.8 mm, length 10 m Reaction reagent flow rate: 0.5 ml/min Measurement results of fluorescence intensity and ultraviolet absorbance
Shown in the figure. As is clear from this, in the former case, the straight line is lost in the vicinity of 3 μg to 4 μg, and the gradient of the straight line becomes large, but on the other hand, the ultraviolet absorption photometry of this invention is characterized by good linearity.

Experimental Example 4 Using glucose aqueous solutions with different concentrations (content 0.4 μg to 7 μg) as samples, ultraviolet absorbance was measured and analyzed under the following conditions, and as in Experimental Example 3, high sensitivity and a good straight line were observed. It was possible to analyze by gender.

Analysis conditions Equipment: Shimadzu high performance liquid chromatograph LC
-3A Analytical column: Shimadzu LC column ISA-07/S2504 Mobile phase: 0.5M - boric acid (adjusted to PH = 9.5 with potassium hydroxide) Mobile phase flow rate: 0.5ml/min Column temperature: 60℃ Ultraviolet absorbance Total: Shimadzu UVD-1 Reaction conditions Reaction reagents: 5.0% taurine - 1/10M, potassium phosphate - 1/20M, sodium tetraborate aqueous solution Reaction temperature: 150℃ Reaction tube: Inner diameter 0.8mm, length 10m Reaction reagent flow rate :0.5ml/min

[Brief explanation of drawings]

Figure 1 is a flow sheet showing an example of an analyzer for carrying out the method of the present invention, and Figures 2 to 5 are chromatograms when sugars are analyzed under various conditions. FIG. 6 is a diagram showing the linearity between peak height and glucose amount when glucose-containing aqueous solutions of various weights are analyzed. 1... Automated saccharide analyzer, 2... High performance liquid chromatography column, 3... Eluent pipe line, 4...
... Reaction reagent supply pipe line, 5 ... Mixing section, 6 ... Heating tank, 7 ... Cooling tank, 8 ... Ultraviolet absorption photometer, 9
...Reaction liquid pipe line, 10...Reaction liquid discharge part, 11...
... pump, 12 ... damper, 13 ... reaction liquid pipe section, 14 ... reaction liquid pipe section, 25 ... chromatography apparatus main body.

Claims (1)

[Claims] 1. A sample containing saccharides is subjected to liquid chromatography, and a reaction reagent of a boric acid aqueous solution containing monoethanolamine or a boric acid buffer solution containing taurine is added to the column eluent, a heating reaction is performed, and then the sample is cooled. , a sugar analysis method characterized by qualitatively or quantitatively analyzing sugars by applying ultraviolet light and measuring its absorbance.