JPS62102141A - Method and instrument for microanalysis of chemical reaction - Google Patents

Abstract

PURPOSE:To make easy, quick and exact measurement of the change with age of the absorption spectra after mixing of a reaction reagent with an intended material with a slight amt. of analytical sample by mixing the reaction reagent liquid with an eluate contg. the intended material of a chromatograph and detecting the change of the absorption spectra thereof with age by a multiwavelength simultaneous detector. CONSTITUTION:The eluates from eluate reservoirs 1a, 1b are so mixed as to attain a prescribed concn. by a gradient device 2 and the mixture is fed to a column 5 for sepn. by a liquid feed pump 3. On the other hand, the sample contg. the intended material injected by a sample injector 4 is conducted to the column 5. The reactive reagent soln. is delivered from a reactive reagent soln. reservoir 7 at a specified flow rate by a liquid feed pump 8 and is contacted with the eluate from the column 5 through a light absorbancy detector 6 by a 3-way joint 9. Both liquids are mixed by a mixing coil 10. The liquid mixture is conducted through a 6-way selector valve 11 to a fluid cell contained in the multiwavelength simultaneous detector 12 and controlled to 0-100 deg.C by which the absorbancy of the liquid mixture is continuously measured.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for microanalysis of chemical reactions and an apparatus therefor.

Conventionally, when a target substance in an analysis sample is separated using a liquid chromatograph and the time-dependent changes of the target substance after mixing with a reaction reagent solution are observed spectrally, the target substance is separated using a liquid chromatograph. After fractionating using the graph, take out the outer tube of the liquid chromatograph, mix the fractionated liquid level reaction reagent solution containing the target substance from this fractionation, make a volume of several ml, and add this mixed solution. Transfer it to a square cell with a capacity of several ml, and use a spectroscopic absorption photometer to measure the change over time in the light absorption intensity at one or several wavelengths, or the change over time in the light absorption intensity in a specific wavelength range. Ta.

However, in this method, the target substance has to be taken out of the liquid chromatograph and mixed with the reaction reagent solution, which is troublesome, and the amount of target substance required to be equal to the capacity of the square cell is required. If the reaction rate is fast, the reaction progresses while the absorption spectrum is being determined, making it impossible to accurately observe the time course of the reaction.

The present invention was made against this background,
The purpose is to provide a method and device that can easily, quickly, and accurately analyze the change in spectrum over time after a target substance in an analytical sample is mixed with a reaction reagent solution using a trace amount of an analytical sample. It's about doing.

In order to achieve the above object, the present invention involves separating a target substance from an analysis sample using a liquid chromatograph, mixing a reaction reagent solution into the eluate containing the separated target substance, and then The mixed liquid is introduced into a micro-capacity flow cell built into a multi-wavelength simultaneous detector, and then held.
The reaction of the target substance in the flow cell is analyzed by observing the change over time in the absorption spectrum of the reaction mixture using a multi-wavelength simultaneous detector. Additionally, the flow cell is temperature controlled to maintain the reaction at a constant temperature.

A suitable apparatus for carrying out the present invention is a separation system using a liquid chromatography method in which a target substance in an analysis sample is separated using a separation column, and a system in which an effluent containing the target substance flowing out from a separation mechanism is circulated. an absorption detector or a multi-wavelength simultaneous detector with a built-in flow cell, a mixing mechanism for mixing a reaction reagent solution into the effluent from the flow cell of the absorption detector or the multi-wavelength simultaneous detector, and a mixing mechanism for mixing the reaction reagent solution with A reaction coil for mixing the effluent containing the target substance, a simultaneous multi-wavelength detector for observing the change over time in the absorption spectrum of the reaction mixture produced in the reaction coil, and and a valve for guiding and holding the reaction mixture to a flow cell containing a simultaneous multi-wavelength detector. There is a place.

An example of this suitable apparatus is shown in FIG. 1 as a flow diagram. What are la and lb in this diagram? 8 syneresis reservoir,
Each contains an eluent for separating a target substance from an analysis sample.

The mixing ratio of these eluents is adjusted by the gradient device 2 so as to have a predetermined concentration gradient, and the eluent is sent to the separation column 5 filled with a packing material used in ordinary liquid chromatography by the eluent supply bomb 13. It is made of camphor so that it can be continuously supplied. A sample injector 4 is provided between the eluent feed pump 3 and the separation column 5, and the sample containing the target substance injected from the sample injector 4 is guided to the separation column 5. ing. Reference numeral 6 denotes an absorption detector or a multi-wavelength simultaneous detector having a built-in flow cell, and continuously monitors the absorbance of the eluate from the separation column 5.

7 is a reaction reagent solution reservoir, and this reaction reagent solution is sent out with a constant flow by a reaction reagent solution feed pump 8, and is also collected from the separation column 5 after passing through an absorption detector or a simultaneous multi-wavelength detector 6. Eluate and three-way joint 9
Both solutions are mixed in a mixer 10 to prepare a reaction mixture. Reference numeral 11 denotes a hexagonal switching valve, which is used to guide and hold the reaction mixture to the flow cell built into the multi-wavelength simultaneous detector 12. That is, in the hexagonal switching valve 11 in FIG. 1, in the case of the flow path shown by the solid line, the reaction mixture is guided to the flow cell built in the multi-wavelength simultaneous detection 12, and in the case of the flow path shown by the dotted line. In case of camphor, the reaction mixture is kept in a flow cell. A temperature control device is connected to the flow cell built into the multi-wavelength simultaneous detection 12 to maintain a constant reaction temperature within the flow cell.

Incidentally, in order to confirm the effects of the present invention, the results of analyzing the time course of the reaction of hemoglobin and glycosylated hemoglobin in mixed hemolyzed samples obtained from diabetic patients and grouped red blood cells patients are shown below.

The measurement conditions are as follows.

Multi-wavelength simultaneous detector: MULTI-320 (manufactured by JASCO Corporation), flow cell volume i-4 μl.

Measurement wavelength - IQ rr from 390nm to 700nm
32 wavelength absorption detector every m: UVIDEC-100-VI (manufactured by JASCO Corporation), flow cell capacity -8μ! ? 8 Syneresis pump and gradient device: TRI
ROTAR-VI (manufactured by E1Bonko Igyo Co., Ltd.) Separation column: DVT-119 (inner diameter 8 mm, length 60 m
(manufactured by Asahi Kasei Industries, Ltd.) Column temperature: 24C Mobile phase: 30mM sodium phosphate vI street solution containing 0.01% sodium azide as solution A (pH 5,50
), 0.01% sodium azide as solution B and 0
．． A 30 mM sodium phosphate MWi solution (pH 5,50) containing 6 M sodium chloride was prepared, the A/B mixing ratio was changed linearly from (89/11) to (87713) in 8 minutes, and then ( 83/17) in 8 minutes, then 75/25 in 8 minutes, then 50150 in 25 minutes, then 6 minutes (50150). ) was retained.

Eluent flow ffi: 1.5 ml/min Reaction reagent solution feed pump: 5P-024 (manufactured by JASCO Corporation) Reaction reagent solution: 0.1M sodium hydroxide aqueous solution Reaction reagent solution flow rate: 1.2 ml/min Reaction temperature = 30
°C Figure 2 shows the chromatogram and representative absorption spectra of a mixed hemolyzed sample from a diabetic patient and a grouped cell patient. Peak 12 is hemoglobin-F, peak 16 is stable glycosylated hemoglobin, and peak 21 is hemoglobin-F. hemoglobin-A
, 24 is hemoglobin-8. Those spec I・
Although only 16 appears to be slightly different, they are almost identical. Figure 3 shows 10.44.7 after mixing with the reaction reagent solution.
8.112. Shows the absorption spectrum change of fractionation 2.16.24 during 359 seconds.

Immediately after mixing with the reaction reagent solution, the spectra of fractions 2 and 24 are almost the same, but the change over time is different from that of fraction 2.
0 is slower and exhibits the characteristics of hemoglobin-F.
Fraction 6, like fraction 24, showed a rapid change in spectrum over time after mixing the reaction reagent solution.

As described above, according to the present invention, it is possible to separate a target substance from a small sample by liquid chromatography, mix it with a reaction reagent solution, and observe the reaction process spectrally.

[Brief explanation of drawings]

FIG. 1 is a flow path diagram showing an embodiment of the apparatus according to the present invention, FIG. 2 is a chromatogram and absorption spectrum obtained in the process of carrying out the method of the present invention, and FIG. 3 is a flow diagram showing an embodiment of the apparatus according to the present invention. This is the time course of the spectrum obtained according to the following. 1a, 1b: Eluent reservoir, 2: Gradient device, 3: Eluent feed pump, 4: Sample injector, 5: Separation column,
6: Absorption detector or multi-wavelength simultaneous detector, 7: Reaction liquid reservoir, 8: Reaction reagent solution feeding pump, 9: Three-way joint,
10: Mixing coil, 11: Six-way switching valve,
12: Multi-wavelength simultaneous detector Fig. 1 Fig. 2 400 500 600 700
nm figure 3

Claims (1)

[Claims] 1) After separating the target substance from the analysis sample using liquid chromatography, a reaction reagent solution is mixed into the eluate containing the separated target substance, and then this mixture is detected at multiple wavelengths simultaneously. The reaction of the target substance in the flow cell is analyzed by guiding the mixture into a flow cell built into the device and holding it, and observing the change in absorption spectrum exhibited by the mixture over time using a simultaneous multi-wavelength detector. An analysis method characterized by 2) An analysis method characterized in that the reaction in the flow cell according to claim 1 is carried out by controlling the temperature in the range of 0°C to 100°C. 3) An analysis method characterized in that the reaction in a flow cell according to claim 1 is carried out in a flow cell with a minute volume of 100 μl or less. 4) A separation mechanism using a liquid chromatography method that separates the target substance in an analysis sample using a separation column, an absorption detector with a built-in flow cell through which the effluent containing the target substance flowing out from the separation mechanism flows, and a light absorption detector. A mixing mechanism that mixes a reaction reagent solution into the effluent from the flow cell of the detector, a reaction coil that mixes these reaction reagent solutions and the effluent containing the target substance, and a reaction generated by the mixing. It is characterized by comprising a multi-wavelength simultaneous detector for observing changes over time in the absorption spectrum exhibited by the mixed solution, and a valve for guiding and holding the reaction mixture into a flow cell built into the multi-wavelength simultaneous detector. Analyzer for 5) A temperature control device for controlling the temperature of a flow cell built into the simultaneous multi-wavelength detector according to claim 4. 6) A capacity of 100 μl for holding the reaction mixture, which is built into the simultaneous multi-wavelength detector according to claim 4.
Flow cell below.