JP3351056B2 - Filler for chemiluminescence detection and analytical method using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a technique for analyzing a substance by a chemiluminescence detection method in liquid chromatography and flow injection.

[0002]

2. Description of the Related Art Conventionally, chemiluminescence reactions include a reaction in which a direct reagent such as luminol or the like itself emits chemiluminescence and a reaction in which a fluorescent reagent indirectly coexisting such as an oxalic acid derivative emits chemiluminescence. ing. In particular, the latter has a high luminous efficiency among the chemiluminescence systems known so far, and hence, analytical research has been actively conducted.

In this reaction, oxalates react with hydrogen peroxide in the presence of a basic catalyst to form an active intermediate,
It is known as a peroxalate chemiluminescence method in which a coexisting fluorescent substance is excited to emit light (MM Rauhut,
D. Sheehan, RA Clarke, and AM Semsel: Photoch
em. Photobiol., 4, 1097 (1965)). In particular, when analyzing and detecting hydrogen peroxide generated directly or through enzymatic reaction using high-performance liquid chromatography, oxalate ester and a fluorescent substance are added to an organic solvent solution to form a reaction solution. A peroxalate chemiluminescence method in which a solution is sent and mixed with an analyte to emit light has been reported (K. Honda, K. Miyaguchi, H. Nishino, H. Tanaka,
T. Yano and K. Imai: Anal.Bio-chem., 153, 50 (198
6) etc.). However, there is a problem in that light emission caused by the reaction between the oxalate ester and the fluorescent substance raises the background and increases the fluctuation of the baseline, so that stable and highly sensitive measurement cannot be obtained. If this background can be suppressed and baseline fluctuation can be reduced, stable and highly sensitive analysis can be performed.

[0004]

SUMMARY OF THE INVENTION In liquid chromatography and flow injection analysis using a chemiluminescence detection method using a peroxalate, the background is reduced, the baseline fluctuation is reduced, and a stable and highly sensitive method is used. It is an object of the present invention to enable the analysis of hydrogen peroxide and peroxide.

[0005]

SUMMARY OF THE INVENTION It has been found that the above object can be achieved by using a filler to which a fluorescent substance according to the present invention is immobilized. That is, the present invention relates to a filler for chemiluminescence detection in which a fluorescent substance is immobilized on a carrier insoluble in a solvent, and further relates to a method for analyzing hydrogen peroxide and peroxide by liquid chromatography using the filler, The present invention relates to a method for analyzing hydrogen peroxide and peroxide by flow injection using the above filler.

[0006] By filling the filler according to the present invention into a flow cell in a chemiluminescence detector and using it, hydrogen peroxide can be directly or via an enzymatic reaction without the presence of a fluorescent substance in the reaction solution. The resulting hydrogen peroxide and peroxide can be quantified stably with high sensitivity.

Here, the fluorescent substance immobilized on the filler according to the present invention generally has fluorescence and emits light when excited by a chemical reaction, and can be directly or an amino group, a carboxyl group, a hydroxyl group, a thiol group. Any compound having a functional group capable of chemically bonding to a carrier, such as an aldehyde group, an epoxy group, or a halogen group may be used. For example, dansyl derivatives, nitrobenzoxadiazole derivatives, coumarin derivatives, perylene derivatives, pyrene derivatives, fluorescein derivatives, fluorescamine derivatives and the like are suitable. In addition, the carrier insoluble in the solvent used for the filler according to the present invention is not particularly limited as long as it is insoluble in water or an aqueous solution containing a salt or an organic solvent, nylon, polystyrene, polyacrylamide, polyacrylate, polycarbonate, Examples include synthetic polymers such as polyesters, polyurethanes, and copolymers and cross-linked products thereof, natural polymers such as cellulose and agarose, and inorganic substances such as silica, alumina, and titania. In addition, the carrier insoluble in these solvents needs to have a functional group such as a hydroxyl group, an amino group, a carboxyl group, an epoxy group, or a halogen for binding the fluorescent substance to the carrier. These may have any shape as long as they can be filled in the flow cell. However, in view of the pressure resistance of the flow cell, it is advantageous that they are spherical with a diameter of several tens μm to several hundred μm. is there.

The immobilization of the fluorophore is not particularly limited, and may be performed directly or as a bifunctional compound such as propanediamine, glutaraldehyde, 1,4-butanediol diglycidyl ether or 4-aminobutyric acid. Can be used as a spacer.
The immobilization reaction is carried out in a suitable solvent according to each functional group in the presence of a catalyst such as an acid, a base or the like, or a condensing agent such as carbodiimides, N-hydroxysuccinimide or carbonyldiimidazole. , Or in the absence thereof according to a conventional method.

The filler according to the present invention obtained as described above is filled in a flow cell in a chemiluminescence detector. The shape of the flow cell used at this time is not particularly limited. Although it may be in the form of a tube, usually a tube made of quartz glass or a fluorinated resin is processed into a spiral type or zigzag to have a total internal volume of 100 to
Those having a volume of about 200 μl are often used to increase the light receiving efficiency (FIG. 1). At this time, the light emission detection method may be either the analog mode or the counting mode.

[0010] Hydrogen peroxide and peroxide using a liquid chromatograph system and a flow injection system (hereinafter, referred to as a system according to the present invention) using a chemiluminescence detector in which a flow cell is filled with the filler according to the present invention. In analyzing the substances, as shown in FIG. 2, the analytes (hydrogen peroxide and peroxide) injected from 13 are eluted by the analytical column by the eluent sent from 11 and post-column. Active intermediate produced by the reaction with the chemiluminescent reagent sent from 17 by the method
The energy generated by the transfer of the energy to the immobilized fluorophore in the flow cell and the return of the excited fluorophore to the ground state is measured in the 15 chemiluminescence detectors. The chemiluminescent reagent used here is one that causes an indirect chemiluminescent reaction, and includes oxalates and the like. As the oxalates, the aryl group of the aryl oxalate is a substituent having a strong electron-withdrawing property (eg, Cl, F, NO
₂ , CF ₃ etc.) are suitable. In particular, bis (2,4,
6-trichlorophenyl) oxalate (TCPO) is a preferred example.

The analysis of hydrogen peroxide is useful not only for general analysis but also especially for analysis of biological substances. At this time, the substance to be analyzed is separated by the separation column, and then converted to hydrogen peroxide by the column on which the hydrogen peroxide-forming enzyme is immobilized and detected. The enzyme to be used is appropriately selected depending on the substance to be analyzed, but depending on the case, it is also possible to detect a finally obtained hydrogen peroxide by combining a plurality of reactions as well as a one-step reaction. There are many reports on immobilized enzyme column technology. For example, reference can be made to Kazuhiro Imai, “Bioluminescence and Chemiluminescence,” p. 233, Hirokawa Shoten (Showa 64) and the like. The analysis of peroxide is also useful in biological substances, food industry, and environmental analysis. As to the analysis conditions when liquid chromatography and flow injection are performed using the system according to the present invention, an eluent containing salts and buffers, a temperature range, a flow rate, and the like which are usually performed can be selected.

[0012]

[Examples] Representative examples of analytical methods using the packing material according to the present invention and the chromatographic system according to the present invention will be described below in more detail. However, these are merely examples for explanation, and it goes without saying that the present invention is not limited to these.

Example 1 The epoxy group of an epoxy-containing gel-like copolymer obtained from glycidyl methacrylate and glycerin dimethacrylate was ring-opened and modified with water, followed by reaction with 1,4-butanediglycidyl ether and further propanediamine. Was dispersed in 2 ml of a 0.1 M aqueous sodium hydrogencarbonate solution to give 7-chloro-4nitrobenzoxadiazole (7. -Chroro-4-ni
2 ml of an ethanol solution to which 30 mg of trobenzoxdiazole was added, and the mixture was shaken at 80 ° C for 20 minutes. The gel was collected by filtration to remove unreacted substances, washed successively with ethanol, distilled water and 2N-hydrochloric acid and dried to obtain a nitrobenzooxadiazole (NBD) derivative-immobilized gel.
The unreacted product was quantified by HPLC, and it was confirmed that 60 μmol / g of nitrobenzoxadiazole was immobilized.

Example 2 The filler obtained in Example 1 was filled in a fluorinated resin tube of a flow cell (inner diameter 1.0 mm, length 600 mm) in a chemiluminescence detector, and a system according to the present invention was used. Various concentrations of aqueous hydrogen peroxide solution were analyzed. As a result, as shown in FIG. 3, a high linear relationship was obtained between the hydrogen peroxide concentration and the chemiluminescence intensity. Analysis conditions Eluent 0.1M Imidazole-NO ₃ (pH7.0) / MeCN (3/1) Flow rate 0.5ml / min 1mM TCPO / AcOEt-MeCN Flow rate 2.0ml / m
in

Example 3 Amino group-containing gel copolymer used in Example 1
0 g was dispersed in 10 ml of 0.2 M NaHCO ₃ , 75 mg of dansyl chloride (DNS-Cl) was dissolved in 10 ml of acetone, and the mixture was shaken at 37 ° C. for 2.5 hours. The gel was filtered to remove unreacted substances, washed successively with acetone and distilled water and dried to obtain a dansylated gel. Unreacted material is H
Quantification by PLC confirmed that 65 μmol / g of dansyl groups had been immobilized.

Example 4 The same procedure as in Example 2 was repeated except that the filler obtained in Example 3 was used in place of the filler obtained in Example 1 to analyze the aqueous hydrogen peroxide solution. As a result, as shown in FIG. 4, a high linear relationship was obtained between the hydrogen peroxide concentration and the chemiluminescence intensity.

Example 5 2 g of the amino group-containing gel copolymer used in Example 1
Tetrahydrofuran (THF) 20 in which 1.0 g of 3,4,9,10 perylenetetracarboxylic dianhydride is dissolved
0.5 ml of pyridine was added to the mixture and shaken for 8 hours. The gel was collected by filtration to remove unreacted substances, washed successively with methanol and distilled water and dried to obtain a perylene derivative-immobilized gel.
The unreacted product was quantified by HPLC, and it was confirmed that 8 mg of the perylene derivative had been immobilized.

Example 6 The same procedure as in Example 2 was repeated, except that the filler obtained in Example 5 was used in place of the filler obtained in Example 1, and an aqueous hydrogen peroxide solution was analyzed. As a result, a high linear relationship was obtained between the concentration of hydrogen peroxide and the intensity of chemiluminescence as in the case of using the gel immobilized with another fluorescent reagent.

[0019]

As described above, the chemiluminescence by the mixed system of the oxalate ester and the fluorophore can be analyzed with high sensitivity, but the background is high and unstable. The filler according to the present invention is obtained by binding a fluorophore to a carrier that is insoluble in a solvent by a stable covalent bond, and can perform measurement without constantly sending the fluorophore. Background noise does not occur and the noise level can be reduced. As a result, stable and highly sensitive analysis of hydrogen peroxide is possible.

[Brief description of the drawings]

FIG. 1 shows an example of the shape of a flow cell.

FIG. 2 is a diagram of a chromatographic system according to the present invention.

FIG. 3 is a graph showing the relationship between the concentration of hydrogen peroxide and the intensity of chemiluminescence when the filler obtained by immobilizing the phosphor obtained in Example 1 is used.

FIG. 4 is a graph showing the relationship between the concentration of hydrogen peroxide and the intensity of chemiluminescence when the filler obtained in Example 3 is used.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Light transmission tube 1A Light transmission tube 1B Light transmission tube 2 Reflecting surface 3 Projection 4 Window plate 5 Cell plate 6 Long groove 6A Long groove 6B Long groove 11 Eluent 12 Pump 13 Injector 14 Column 15 Chemiluminescence Detector 16 Recorder 17 Chemiluminescent reagent solution

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification symbol FI G01N 30/88 G01N 30/88 H JP, A) JP-A-63-75540 (JP, A) JP-A-60-149950 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 30/48 G01N 21/76 G01N 30/74 G01N 30/88

Claims (5)

(57) [Claims] 1. A method for immobilizing a fluorescent substance on a carrier insoluble in a solvent.
Use of peroxalate.
Chemiluminescent detection filler for that. 2. It is characterized by being immobilized by a covalent bond.
The filler for chemiluminescence detection according to claim 1. 3. The method according to claim 1, wherein the fluorescent substance is a dansyl derivative, nitroben
Zoxadiazole derivative, coumarin derivative, perylene
Derivative, pyrene derivative, fluorescein derivative or
3. The fluorescamine derivative according to claim 1 or 2,
Filler for chemiluminescence detection. 4. The analysis method according to claim 1 to hydrogen peroxide and peroxide by liquid chromatography using a filler according to any one of the three. 5. A method for analyzing hydrogen peroxide and peroxide by flow injection using the filler according to any one of claims 1 to 3 .