JP4909234B2 - Method for detecting substances to be measured by electrolysis - Google Patents

Description

  The present invention provides a method for detecting a component of a substance to be measured in an aqueous solution by performing electrolysis with an electrolytic device provided in a flow path of the aqueous solution. By using an electrolytic device having a specific configuration using a diamond electrode, a trace amount can be obtained. The present invention relates to a method for detecting various organic compounds.

  One method for detecting components in a solution containing various organic compounds is to measure the electrolytic current of the solution. Carbon-based materials, metals, metal oxides, semiconductors, and the like are known as electrodes used for such electrochemical detection, and particularly carbonaceous electrodes such as glassy carbon are widely used.

  Further, as disclosed in Japanese Patent Application Laid-Open No. 11-83799 (Patent Document 1), a diamond electrode has attracted attention as a working electrode of an electrolysis apparatus. In Patent Document 1, a diamond electrode in which a diamond thin film is formed on a silicon single crystal substrate by plasma-excited CVD is used. Note that a small amount of boron is mixed to provide conductivity. Then, such a detection electrode made of diamond (the working electrode is referred to in this way in Cited Document 1), a counter electrode and a reference electrode are held side by side in the electrolytic cell, and a voltage is applied between the electrodes to measure the electrolysis current. is doing.

In Patent Document 1, it is assumed that the concentration of each component can be obtained in a solution in which a plurality of components are mixed by utilizing the fact that the voltage at which electrolysis starts depends on the substance to be measured. For example, on a diamond electrode, glucose oxidation begins at + 2.2V and ascorbic acid oxidation begins at + 0.7V. Therefore, for example, if electrolysis is performed at +1.5 V and +2.5 V and the response current at that time is obtained, the response current at +1.5 V corresponds to only ascorbic acid, and the response current of +2.5 V corresponds to the sum of ascorbic acid and glucose. Therefore, the respective concentrations can be obtained. As the current, a value of 0.5 mA / cm ² is shown at +2.5 V when the glucose concentration is 5 mM.

  In Cited Document 1, since the electrolytic cell is filled with a solution having a concentration of 5 mM as in the above example, the total amount of the substance to be measured is considerably increased. Even if the concentration is low, the amount of the substance to be measured is small if it can be measured. However, the detected current value is also low, and the measurement is difficult due to the influence of disturbance. Cited Document 1 shows an electrolysis apparatus in which an electrode is inserted into an electrolytic cell, and a reference electrode, a detection electrode, and a counter electrode are arranged in a row with an electric insulating layer interposed therebetween. Since the measurement is performed in a state where the electrolyte containing the substance to be measured is in contact with one surface of each electrode, there is a possibility that the amount of the electrolyte can be reduced as compared with the case of the electrolytic cell. Examples of actual measurements are not shown.

  On the other hand, Japanese Patent Laid-Open No. 2001-50924 (Patent Document 2) shows an apparatus to which a diamond electrode is applied in an electrolysis apparatus generally called a flow cell that continuously introduces a sample solution and discharges it after performing electrolysis. ing. In Cited Document 2, in the drawing showing the electrolysis apparatus, only three lines indicating the counter electrode, the working electrode, and the reference electrode are arranged in parallel in a quadrilateral, and there is no detailed explanation about the structure. A flow injection analysis is shown in which an electrolytic current is measured by intermittently injecting a sample solution in a state where a buffer solution is continuously passed through such a flow cell.

For example, with a set potential of 1.275 V (vs. Ag / AgCl), a 0.1 M phosphate buffer solution is allowed to flow at a flow rate of 1 ml / min, and 20 μl of a solution containing histamine at each concentration ranging from 1 μM to 100 μM. The electrolytic current is measured by injection. As a result, a peak current was confirmed each time it was injected, and in the case of 100 μM, it was 437.096 nA. In a solution having a lower concentration than this, a low current proportional to the concentration is obtained, so that accurate concentration measurement is possible.
Japanese Patent Laid-Open No. 11-83799 JP 2001-50924 A

  Electrolysis using a flow cell such as the flow injection analysis shown in the cited document 2 can separate and quantify each component in a mixed solution when combined with liquid chromatography. Moreover, since the diamond electrode has a wide potential window and water electrolysis does not occur even at a considerably high voltage, the electrolysis current can be detected in a wide range of compounds. However, in the analysis method of the cited document 2, the peak value of the current detected even when the sample concentration is 100 μM is about 0.4 μA. According to the description of the cited document 2, in general, in the analysis using the flow cell, the pulsation greatly influences the analysis. However, in the conductive diamond electrode, the background current is extremely small, so the influence of the pulsation is extremely small, and the accurate analysis is possible. It is possible. However, the electrolysis current is weak in a medical test sample or the like in which the concentration of a measurement target substance such as measurement of a trace component in blood is extremely low, and the sensitivity of the measurement method of the cited document 2 is insufficient. An object of the present invention is to provide a method for detecting a very small amount of a substance with high sensitivity in the case of performing analysis using an electrolysis apparatus using a diamond electrode due to the above problems.

The present invention solves the above-mentioned problem, and in a method for detecting a component to be measured in a sample solution by performing electrolysis with an electrolytic device provided in a flow path of the sample solution, the electrolytic device is an electrode of a conductor. A working electrode having a conductive diamond film formed on at least one surface of the plate, a counter electrode made of a conductor facing the surface having the diamond film of the working electrode with an interval of 0.05 to 1.0 mm, and a working electrode at the tip And a reference electrode facing each other with an interval of 0.05 to 1.0 mm, and a portion of the working electrode having a diamond coating facing the counter electrode and the reference electrode and serving as a flow path for the sample solution width be one which is the 1.5mm or 4.0mm or less, the voltage between the working electrode and the counter electrode while flowing the sample solution between the working electrode and the counter electrode and a reference electrode A method for detecting a substance to be measured by electrolysis, characterized in that the concentration of the substance to be measured is measured by applying and generating an electrolytic current of 0.1 μA or more per 1 μM concentration of the substance to be measured and measuring the electrolytic current. is there. Here, it is also characterized in that the electrolysis current is due to the breakage of the bond between the atoms of the molecule.

  According to the method for detecting a substance to be measured by electrolysis of the present invention, by using conductive diamond having a large potential window as a working electrode in an electrolysis apparatus, electrolysis of water occurs due to high electrolysis voltage, and carbonaceous matter is generated. This makes it possible to detect a substance that cannot be detected by this electrode. Furthermore, by making the distance between the working electrode and the counter electrode and the distance between the working electrode and the reference electrode as extremely narrow as 0.05 to 1.0 mm, an electrolytic current that can be sufficiently measured even with a very small amount of components can be obtained.

  In the present invention, electrolysis is performed by an electrolysis apparatus provided in a flow path of an aqueous solution to detect a substance component to be measured in the aqueous solution. The present invention is applied to flow injection analysis in which a solution is put in, post column analysis in which an eluent flowing out from a liquid chromatograph is introduced into an electrolysis apparatus, and the like. The electrolysis apparatus used in the present invention uses conductive diamond as a working electrode, and it is possible to generate a large electrolysis current with a small amount of components by specially arranging the counter electrode and the reference electrode. I have to.

  In the method of the present invention, by using a diamond electrode as described above as a working electrode of an electrolysis apparatus for detecting a substance to be measured, it is possible to perform electrolysis by applying a higher voltage than before. That is, when a carbonaceous electrode such as glassy carbon generally used as a working electrode in a conventional electrolysis apparatus has an anode potential exceeding 1.2 V with respect to a standard hydrogen electrode (hereinafter referred to as “NHE”), Decomposition occurs, making it difficult to measure the electrolysis current due to the substances in the components. On the other hand, the electrolysis apparatus used in the present invention does not cause electrolysis of water up to about 2.5 V (relative to the Ag / AgCl reference electrode). Therefore, in the method for detecting a substance to be measured according to the present invention, since the electrolysis voltage is high, it is possible to detect a substance that could not be detected conventionally.

  1 and 2 are views showing an electrolysis apparatus used in the present invention. FIG. 1 is a cross-sectional view parallel to the axial direction, and FIG. 2 is a cross-sectional view taken along line AA ′ in FIG. In these figures, reference numeral 11 denotes a working electrode (in FIG. 2, the positional relationship is indicated by a two-dot chain line), which is at least on the front surface of the thin plate-like conductive substrate, that is, on at least the left surface in FIG. A conductive diamond film is formed. Reference numeral 12 denotes a spacer made of a chemical-resistant electrical insulator such as fluorine resin, and has one elongated hole 121 as shown in FIG. Reference numeral 13 denotes a counter electrode made of a corrosion-resistant conductor block such as titanium, which faces the surface of the working electrode 11 on which the diamond is formed with the spacer 12 interposed therebetween.

  The working electrode 11 is a conductive thin plate, for example, a conductive silicon single crystal plate having a thickness of about 0.7 mm, and has a thickness of about 30 μm made of fine diamond crystals of several μm in size. Created by forming a film. The diamond film can be formed by plasma CVD in hydrogen gas containing a carbon source such as acetone. In order to impart conductivity to diamond, boron oxide or the like is doped in boron by dissolving it in the carbon source.

  There are an elution inlet 15 and an outlet 16 and a reference electrode 30 respectively opened in one space 14 formed by the hole 121 of the spacer 12, all of which are provided in the block of the counter electrode 13. Yes. That is, the eluate inlet 15 and the outlet 16 are formed by making holes in the counter electrode 13 block, and fluid couplings 17 and 18 for introducing and discharging the eluate are screwed into the counter electrode block. The reference electrode 30 is also screwed into the counter electrode block. Reference numeral 19 denotes an energization terminal for the counter electrode 13.

  The reference electrode 30 is an example of an Ag-AgCl system. However, a chemical resistant container 301 such as a fluororesin in which a saturated KCl solution is gelatinized with gelatin is filled as an electrolytic solution 302. . Further, an Ag wire having a surface made of AgCl is inserted as an electrode material 303 therein. Reference numeral 304 denotes a filter made of porous ceramic or the like that separates the electrolyte solution 302 of the reference electrode from the eluate that is the liquid to be measured.

  Further, a current-carrying plate 20 for the working electrode is provided on the back surface of the electrode plate of the working electrode 11, that is, on the opposite side of the surface facing the counter electrode 13, and is in contact with the working electrode 11. Reference numeral 21 denotes an energization terminal for the working electrode 11. Reference numeral 22 denotes a pressurizing cover made of a chemical-resistant electrical insulator, which is coupled to the block of the counter electrode 13 by a plurality of set screws (not shown), and is sealed with O-rings 23 and 24 so as to be electrolyzed. Is kept in a pressurized state. In addition, an O-ring 26 is provided in a part of the energization plate 20 so that the sample solution does not enter inside thereof, and the energization plate is directly brought into electrical contact with the back surface of the electrode plate without a liquid. A plastic insulating cover 27 covers the entire metal block of the counter electrode 13.

As described above, the inside of the electrolysis apparatus is in a pressurized state, but the influence of electrolysis of water on the back surface and the end surface of the electrode plate of the working electrode 11 where the diamond is not formed and on the surface of the energizing plate 20 is affected. This is to reduce it. That is, since it is difficult to seal the liquid on the surface of the electrode plate of the working electrode due to the problem of damage to the electrode plate due to the applied pressure, the electrolysis apparatus according to the present invention also includes the back surface and the end surface of the electrode plate of the working electrode 11 with the sample solution. It has a structure that allows contact. For this reason, gas is generated by electrolysis of water on the back and end surfaces of the working electrode, the surface of the energizing plate, and the like. However, by pressurizing the inside of the electrolysis apparatus, the generated gas film can be stably maintained, and when the apparatus is started and the time has elapsed, the electrolysis can be stably stopped at these points. Therefore, it is possible to prevent the electrolysis current of water from flowing from time to time during analysis and causing disturbance to the measured value. The applied pressure is suitably 1.0 kg / cm ² or more and 15.0 kg / cm ² or less (gauge pressure) in view of the effect and the problem of sealing of the apparatus.

  Furthermore, in the electrolysis apparatus of the present invention, the distance between the working electrode and the counter electrode is set to 0.05 to 1.0 mm. Since this interval is regulated by the thickness of the spacer 12, the thickness may be adjusted. Furthermore, not only the distance between the working electrode and the counter electrode, but also the distance between the reference electrode and the counter electrode is set to 0.05 to 1.0 mm. Since the reference electrode is screwed and attached to the counter electrode block as described above, the distance between the reference electrode and the counter electrode depends on the reference electrode mounting position and the distance between the working electrode and the counter electrode. It is maintained at a predetermined value. If the tip of the reference electrode is made the same as the position of the electrode surface of the counter electrode, both the counter electrode and the reference electrode have the same spacing as the working electrode, but this is usually the case.

  In this way, by making the working electrode 11 and the reference electrode 30 and the electrode surface of the counter electrode 13 face each other with a narrow gap, an extremely large electrolysis current can be obtained with a small amount of components. That is, even if either the distance between the working electrode and the counter electrode or the distance between the reference electrode and the counter electrode is larger than the above, an electrolysis current is observed, but as shown in the cited reference 2, it is detected with a sample having a concentration of 100 μM. The applied current is about 0.4 μA. However, in the electrolysis apparatus of the present invention, an electrolysis current 100 times or more can be obtained with the same component amount. Therefore, in the electrolysis apparatus according to the present invention, it is possible to obtain a current that can be detected even by a much smaller amount of sample than in the method of Reference 2. Summing up the experimental results of various organic compounds, in the present invention, an electrolysis current of 0.1 μA or more per 1 μM (1 pmol / μl) concentration of the substance to be measured in the solution is obtained. Therefore, if there is a concentration of about 1 μM, the concentration of the substance to be measured can be measured by measuring the electrolysis current.

  If the distance between the working electrode and the reference electrode and the counter electrode is smaller than 0.05 mm, the working electrode or the reference electrode and the counter electrode may come into contact with each other. There is. More preferable in this respect is 0.5 mm or less. At this time, the width W (FIG. 2) of the surface of the working electrode having the diamond coating facing the counter electrode and the reference electrode and serving as the aqueous solution flow path is 1.5 mm or more and 4.0 mm or less. Is preferred. That is, as described above, when the distance between the working electrode and the counter electrode was narrowed, the variation in the electrolysis current value was observed. Therefore, a model was made of a transparent plastic and the flow condition was examined. As a result, it was found that when the width of the flow path between the working electrode and the counter electrode is wide, the flow branches in a streak shape, and the electrolysis current value fluctuates due to movement. Therefore, the flow width is prevented from changing in the width direction by reducing the width of the flow path to 4.0 mm or less. On the other hand, even if the width of the flow path is smaller than 1.5 mm, the above effect is not increased, and only the electrolytic current is reduced.

The weak electrolysis current as shown in the cited document 2 is considered to be due to electron detachment, that is, ionization. On the other hand, it was found that the reason why an extremely large electrolysis current can be obtained with a very small amount of components in the electrolysis apparatus according to the present invention is due to a change in molecular structure, in particular, a break in bonds between atoms of molecules. This means that it is not just an oxidation reaction. For example, in a normal chemical reaction, oxidation of ethanol C ₂ H ₅ OH results in acetaldehyde CH ₃ CHO, and further acetic acid CH ₃ COOH. Become water. However, in the electrolysis apparatus of the present invention, it has been found that hydroxyl groups are detached in the case of ethanol.

In this way, it was also observed in other compounds that the bond between the atoms of the molecule was partially broken. For example, cleavage of an amino group (—NH ₂ ), carboxyl group (—COOH), or thiol group (—SH), or cleavage of an ester bond or ether bond occurs. It has also been found that bond breakage occurs not only between these functional groups but also between chain-bonded atoms. For example, in the case of organic compounds having cyclic bonds such as benzene and naphthalene in addition to ethane, butane, propane, etc., the molecular bond is broken. Furthermore, it was confirmed that molecular bonds were broken in organic halogen compounds such as polychlorinated biphenyls and chlorobenzene. Further, the breakage of the bond between the atoms of the molecule is not necessarily one place, and in a complex compound, the breakage may occur at a plurality of places at the same time. Since the electrolysis current generated with the breakage of the bond between atoms of these molecules is proportional to the concentration in each molecule, the component concentration of the solution can be measured by measuring the current.

  The reason why the large electrolysis current associated with the breaking of the bond between the atoms of the molecule as described above is generated only when the working electrode, the reference electrode, and the counter electrode are opposed to each other with a narrow gap of 0.05 to 1.0 mm. It is unknown. However, in view of the phenomenon, it is estimated that a large potential gradient by narrowing the interval and a change in the structure of the electric double layer due to this are involved. However, since the current does not flow in the reference electrode itself, it cannot be inferred why the tip of the reference electrode needs to face the working electrode with a narrow space.

Example 1
Electrolysis was performed with the electrolysis apparatus shown in FIGS. 1 and 2 to detect the component of the substance to be measured in the sample solution. The distance between the working electrode 11 and the counter electrode 13 was changed to 0.2 mm, 0.5 mm, 1.0 mm, and 2.0 mm by using an electrolysis apparatus having spacers 11 having different thicknesses. In addition, since the tip position of the reference electrode 30 is the same as the electrode surface of the counter electrode in any case, the distance between the reference electrode and the counter electrode is also the same as the distance between the working electrode and the counter electrode. The width of the channel on the surface of the working electrode is 2.5 mm in any case. The working electrode applied voltage of the electrolysis apparatus was set to 2.2 V (the same applies to the Ag / AgCl reference electrode), but no water electrolysis occurred after the power was turned on and the steady state was reached. .

While supplying a buffer solution of 100 mM-KH ₂ PO ₄ solution to the electrolyzer at a rate of 0.5 ml / min, a sample with a concentration of 1 pmol / μl (concentration 1 μM) of ethanol was placed in front of the 20 μl electrolyzer. When injected into the flow channel, when the distance between the working electrode and the counter electrode is 0.2 mm, 0.5 mm, and 1.0 mm, peaks of electrolytic current of 2.4 μA, 2.0 μA, and 1.7 μA are detected, respectively. It was. However, in the case of 2.0 mm, although a slight electrolysis current was recognized, the electrolysis current value was difficult to measure due to the background current.

  Further, in the case where the distance between the working electrode and the counter electrode is 0.5 mm, a sample was injected with the ethanol concentration changed to 10 pmol / μl, 100 pmol / μl, 1 nmol / μl in addition to the above 1 pmol / μl. An approximately proportional electrolysis current was obtained. The injected sample solution is diluted by mixing with the buffer solution at the front and rear end positions of the flow progress, but it is considered that the injected sample solution reaches the electrolysis apparatus at the central position of the flow progress.

(Example 2)
In the electrolysis apparatus used in Example 1, electrolysis was performed by changing the conditions for the reference electrode, and the component of the substance to be measured in the sample solution was detected. However, the distance between the working electrode and the counter electrode was kept constant at 0.5 mm. As condition A, the attachment position of the reference electrode 30 with respect to the counter electrode 13 was changed, and the tip portion was retracted 1.5 mm from the electrode surface of the counter electrode. Therefore, when the distance between the working electrode and the counter electrode is 0.5 mm, the distance between the reference electrode and the counter electrode is 2.0 mm. As condition B, the position of the tip of the reference electrode is the same as the electrode surface of the counter electrode, but the electrical circuit is disconnected from the reference electrode, and another reference electrode is provided in the inflow path of the sample solution to the electrolyzer instead. An electric circuit was connected to this. In this case, the reference electrode attached to the counter electrode can be regarded as a mere insulator wall.

In the same manner as in Example 1, 2.2 V was applied to the working electrode, and a 100 mM KH ₂ PO ₄ solution buffer was supplied to the electrolysis apparatus at a rate of 0.5 ml / min, and ethanol was 1 pmol / μl. A sample with a concentration of 20 μl was injected into the flow path in front of the electrolyzer. However, in either case of condition A or condition B, no electrolysis current detectable with the same measurement sensitivity as in Example 1 was detected.

Example 3
In the electrolysis apparatus used in Example 1, electrolysis was performed by changing the conditions regarding the width of the flow path at the working electrode. That is, the width of the portion of the working electrode having the diamond coating facing the counter electrode and the reference electrode and serving as the flow path for the sample solution is the same as that in the first embodiment by changing the shape of the hole 121 of the spacer 11 2. In addition to .5mm, it was changed to 3.5mm and 5.0mm. The intervals between the counter electrode and the reference electrode and the working electrode were all 0.2 mm.

In the same manner as in Example 1, 2.2 V was applied to the working electrode, and a 100 mM KH ₂ PO ₄ solution buffer was supplied to the electrolysis apparatus at a rate of 0.5 ml / min, and ethanol was 1 pmol / μl. A sample with a concentration of 20 μl was injected into the flow path in front of the electrolyzer. When this is repeated 20 times for each condition of the flow path, the fluctuation range of the peak value of the electrolysis current is 25 nA when the flow path width at the working electrode is 2.5 mm, and 31 nA when it is 3.5 mm. there were. On the other hand, it is 260 nA at 5.0 mm, and the peak value of the electrolysis current is 2.4 μA as described in Example 1, so that it reaches 10% of this and causes an analysis error.

Sectional view parallel to the axial direction showing the electrolyzer used in the present invention AA 'arrow sectional view in FIG.

Explanation of symbols

DESCRIPTION OF SYMBOLS 11 Working electrode 12 Spacer 121 Hole 13 Counter electrode 14 Space 15, 16 Elution liquid introduction port and discharge port 17, 18 Fluid coupling 19 Current terminal 20 Current plate 21 Current terminal 22 Pressure cover 23, 24, 26 O-ring 27 Insulation cover 30 Reference electrode 301 Container 302 Electrolyte 303 Electrode material 304 Filter

Claims (2)

In a method for detecting a component to be measured in a sample solution by performing electrolysis with an electrolysis device provided in a flow path of the sample solution, the electrolysis device has a conductive diamond film formed on at least one surface of a conductive electrode plate. The working electrode, a counter electrode made of a conductor facing the surface of the working electrode having a diamond coating with a spacing of 0.05 to 1.0 mm, and a tip facing the working electrode with a spacing of 0.05 to 1.0 mm The width of the portion of the surface having the diamond electrode coating of the working electrode facing the counter electrode and the reference electrode and serving as the flow path for the sample solution is 1.5 mm or greater and 4.0 mm or less. It is those who are, 0 per concentration 1μM of substance to be measured by applying a voltage between the sample solution working electrode and the counter electrode while flowing between the working electrode and the counter electrode and a reference electrode To generate more electrolytic current 1 .mu.A, the detection method of a measured substance by electrolysis, characterized by measuring the concentration of an analyte by measuring the electrolysis current. 2. The method for detecting a substance to be measured by electrolysis according to claim 1, wherein the electrolysis current is caused by breaking bonds between molecular atoms.

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