JPS58225351A - Analytical apparatus - Google Patents

Abstract

PURPOSE:To enhance analytical preciseness, by providing a removing apparatus due to a dialytic principle for removing or reducing a component not suitable for detecting an analytically objective component present in an elution liquid between a separation column and a detector. CONSTITUTION:The elution liquid in an elution liquid tank 1 is flowed so as to be passed through a specimen injector 3, a separation column 4, a removing apparatus 5, an immobilized enzyme column 8 and a detector 9 by a pump 2 and, at the same time, the reaction liquid in a reaction liquid tank 6 is sent by a pump 7 to be combined with the elution liquid stream at a point before the immobilized enzyme column 8. The removing apparatus 5 has such a structure that a hollow pipe 51 having dialyzability is immersed in a solution having a composition prepared by removing a removal objective component considered not suitable for detection from the composition of the elution liquid and the removal objective component is penetrated and diffused into the solution from the pipe wall according to a dialytic principle during the passage of the effluent liquid from the separation column 4 through the hollow pipe 51.

Description

[Detailed description of the invention] The present invention relates to an analysis device.

When analysis is performed using an analyzer using a separation column such as a liquid chromatograph, for example, non-proteins such as bile acids in serum, steroid compounds such as androsterone, plutisol, pregnanediol, and estrogen, and amines such as catecholamines and polyamines are analyzed. In order to accurately separate these components in a separation column when quantifying blood concentrations of components and various drug substances, the eluent contains specific organic solvents, salts, surfactants, and 2-mercaptoethanol. Additives are added to the eluent to improve separation when the flow from the separation column is passed through a detector to detect the target component in the flow. Additives may adversely affect the detection. In other words, when the effluent is passed through an immobilized enzyme column and the target substance is replaced by a substance that can be detected by a detector, such as a fluorescence detector, by the action of the enzyme, the specific organic The solvent may deactivate the immobilized enzyme, and when detection is performed using ultraviolet or visible light absorption of the target component, the absorption of the added component in the eluent and the absorption of the target component In addition, when detecting using the difference in refractive index between the target component and the eluent, KVi, eluent containing additives added to improve separation in the elution column may overlap. There are cases where the refractive index of the liquid approaches the refractive index of the component to be analyzed, making detection difficult.

In a case like this, the separation column maintained good separation accuracy without changing the composition of the eluent, and the detection accuracy was reduced by the detection inhibitor contained in the eluent in the detection system seconds after the separation column. It was developed with the aim of providing an analyzer that can detect target components without reducing the A hollow tube having dialysis properties is provided, and a removal device is provided for removing and reducing components undesirable for detection of the analysis target component present in the column, and the separation column effluent flows through the removal device. The analyzer is immersed in a solution having a composition in which the above-mentioned undesirable components are removed from the eluent.

The apparatus of the present invention will be explained below with reference to the drawings.

Figure vJ1 is a system diagram showing one embodiment of the device of the present invention.

1 in the figure is an eluent tank, and the eluent in tank 1 is pumped 2.
At the same time, the reaction solution in the reaction solution tank 6 is passed through the sample injector 3, the separation column 4, the removal device 5, the immobilized enzyme column 8, and the detector 9. It is arranged to be combined with the eluent stream before the immobilized enzyme column 80. In an analysis system in which a reaction solution and an immobilized enzyme 8 as shown in the figure are used, the components to be analyzed are separated by a separation column 4 that is active for a detector such as a fluorometer. This is a system that is used when the sample cannot be detected by the detector 9.The effluent from the separation column 4 is led to the identification enzyme column 8, where it is already separated from the target component by the action of the enzyme. By reacting with the reactant contained in the combined reaction solution, the target component for analysis is transformed into a detectable substance, or a detectable substance is generated in an amount corresponding to the amount of the target component for analysis. This system makes it possible to quantify the component to be analyzed using the detector 9 in seconds. It is also a record 11 for recording the detection by the 10#'i detector 9.

An example of a component to be analyzed using the above-mentioned analysis system is each component of bile acids. Column, N
Using a reaction solution containing AD+ (cotinamide adenine dinucleotide) and a fluorometer as a detector, the bile acid component and NAD were reacted by the action of 30+-1 (SD) in an immobilized enzyme column, and as a result, Analysis can be performed by using a detector to detect the fluorescent substance NAD) 1 produced in an equal amount to the reacted bile acid.

However, in general, the enzyme activity of immobilized enzyme columns is impaired by organic solvents in the eluent, such as acetonitrile in bile acid analysis, which are necessary to improve the separation accuracy in the separation column, making long-term analysis difficult. In the present invention, in order to reduce by 1 ton of such components that are necessary for separation using the separation column 4 but are undesirable for subsequent detection of the target component, a removal device is used. fft5 is provided at the same time as the separation column 4 and the detector 8. The five removal devices have a structure in which a hollow tube 51 having dialysis properties is immersed in a solution having a composition in which the target component to be removed, which is considered undesirable in detection, is removed from the eluent composition. The ends 52 and 53 are connected to the analysis system so that the effluent from the separation column 4 passes through the hollow tube 51. Then, the above-mentioned effluent flows into the hollow tube 5
According to the principle of dialysis, components contained in the eluent but not contained in the solution, that is, the target components to be removed, permeate into the solution from the wall of the hollow tube 51 and dissipate. As a result, the component to be removed is removed or reduced from the separation column effluent flowing through the hollow tube 51. Therefore, the hollow tube 51 used is one that has dialysis properties against the target component to be removed, but it also needs to be protected from being attacked by the solution it comes into contact with.
It is required to be able to withstand the internal pressure applied to the system iK, and to be inert and not adsorbed to the substance to be analyzed. Further, it is also possible to use a bundle of a plurality of thin tubes having dialysis properties as the hollow tube 51.

Further, as the hollow tube 51, it is also possible to use a tube that has both dialysis properties and ultra-RI permeability for the component to be removed.

The hollow tube 51 suitable for use in the present invention includes a dialyzable tubular body made of a polymeric material such as regenerated cellulose, cellulose acetate, polyacrylonitrile, polysulfone, or polypropylene. It is also possible to use thin tubes commercially available as kidney dialysis membranes.

In the following, we have explained the case where an immobilized enzyme column is used, such as in the analysis of bile acids, etc. However, even when an immobilized enzyme column is not used, as is limited to the device of the present invention, it is possible to The light absorption or refractive index of the contained components is similar to that of the target component, making it difficult for the detector to detect the target component, or other components in the eluent are undesirable at the stage of detecting the target component. If removing the components in the eluent by showing the behavior of the eluent gives good results in the analysis, it can be adopted, and excellent effects can be expected.

The present invention is an apparatus as described above, and in particular, between the separation column and the detector, it is necessary to separate the target component for analysis by the separation column contained in the separation column effluent.
In addition, in the subsequent stage of detection of the components to be analyzed, a removal device with a specific structure is installed to remove the undesirable components present, so that the undesirable components can be effectively removed. It is excellent to eliminate the adverse effects of the component on detection by removing it, for example, to improve the accuracy of analysis, or to prevent a decline in the detection ability of the detection system and enable long-term continuous analysis. Therefore, it can have a great effect.

Next, examples of the present invention will be described.

Example 1 Bile acids extracted from human serum were repeatedly analyzed using the apparatus shown in FIG.

Here, Micropondano (trade name) manufactured by Quarters Co., Ltd. was used as the separation column 4, and an IJ fluorometer was used as the detector 9, and measurement was performed at an excitation wavelength of 365 nm and a fluorescence wavelength of 450 nm.

As the removal device 5, a hollow fiber for artificial kidneys made of regenerated cellulose (manufactured by Enka Co., West Germany, cut into a length of 2 m) 5 made of wood bundled together is used as a hollow tube 6. For the immobilized enzyme column 8, cellulose fine particles (a diameter of about 80 μ
), the enzyme 3α-ISD is added to the acetonitrile in which cyanide pseudo-f7f is dissolved in sodium subacid aqueous solution H (! tv a-s) to activate the surface of the microparticles, and 3α-ISD is added to the microparticles. A column having a length of 100 m and an inner diameter of 4 fl was filled with 3α-HSD-immobilized cellulose fine particles that had been immobilized by adding and stirring a carbonate buffer solution in which 3α-HSD was dissolved.

11! Using an aqueous solution containing 300 me acetonitrile and L59 diammonium phosphate as an eluent,
The eluent is transferred from the eluent tank 1 to the separation column 4, the removal device 5, and the immobilized enzyme column 8 at a flow rate of 1 m/min by the pump 2.
A 0.026 M birophosphoric acid mild lrJ solution (P H9 , 5) at a flow rate of 111,127 min by pump 7 to the immobilized enzyme column 8
The eluent stream was then merged with the eluent stream before entering.

When the bile acid sample was injected from the sample injector 3, it was fractionated in the separation column 4. Then, repeat the analysis for 500 hours yi, t.

Even when it was carried out, there was almost no inactivation due to the acetonitrile contained in the eluted Nt solution of identification enzyme column 8, and the same activity as the first one was shown. It was confirmed that about 90% of the particles were removed when the particles passed through the removal device 5 (17).

Comparative Example 1 Bile acid analysis was continuously repeated in the same manner as in Fip Example 1 except that the removal bag WII5 was not used. The identified enzyme was gradually inactivated by acetonitrile in the eluent, and After hours

[Brief explanation of drawings]

FIG. 1 is a system diagram showing an example of the apparatus of the present invention. 1...W# syneresis tank, 2.7...pump, 3...sample injector, 4...separation column, 5...removal device, 6...hollow tube with dialysis properties , 6... Reaction liquid tank, 8... Immobilized enzyme column, 9... Detector, 10
...Stirrup meter patent applicant Mototoshi Fujinuma, representative of Sekisui Chemical Co., Ltd.

Claims (1)

[Claims] t. A removal device is provided between the separation column and the detector to remove or reduce components that are present in the separation column effluent and are undesirable for detection of the analysis target component,
The removal device is characterized in that a hollow tube having dialysis properties through which the separation column effluent flows is immersed in a solution having a composition in which the above-mentioned undesirable components are removed from the eluent. Device.