JPH0752175B2 - Electrochemical detector - Google Patents

Description

TECHNICAL FIELD The present invention relates to an electrochemical detector for high performance liquid chromatography.

(Prior Art) An electrochemical detector (ECD) is used as one of detectors for a substance to be measured in high performance liquid chromatography. The ECD is a detector that performs quantification by measuring the current generated by changes (oxidation, reduction, etc.) in the measured substance due to electrochemical reactions. This ECD is composed of a cell having a working electrode, a counter electrode and a reference electrode, and a detector. As the working electrode of ECD, glassy carbon obtained by molding graphite carbon at a pitch and sintering it at around 2000 ° C is widely used. Glassy carbon has high hardness, excellent chemical stability, and high sensitivity when used as an electrode. However, when the detection sensitivity is increased to measure a minute amount of the substance to be measured (for example, when the measurement is performed at a high potential of 1000 mV or higher), the noise level increases, and thus the sensitivity is substantially reduced. The cause is not clear, but it is considered that the surface part of the glassy carbon electrode is eroded and partially collapsed due to the application of voltage. Further, since the glassy carbon is obtained by sintering at a high temperature of around 2000 ° C as described above, variations (density, uniformity, etc.) occur among lots due to subtle differences in the conditions.

On the other hand, a solid potential (polymer-carbon electrode) formed by adding a synthetic polymer compound as a binder to graphite carbon has been proposed. Examples of the polymer compound (polymer) used include Kel-F (manufactured by Daiflon), Teflon (registered trademark), polypropylene, and styrene-divinylbenzene copolymer. These polymer-carbon electrodes have an advantage that the background current (BG, C.) value is smaller than that of the above-mentioned glassy carbon, and thus the background noise level is low. The BGC value is lower as the polymer content in the composition of the electrode is higher. However, the higher the polymer content, the lower the absolute sensitivity. Generally, the working electrode for ECD is evaluated by the sensitivity during measurement, the conductivity of the electrode itself, and the BGC value. It is desirable to develop an ECD that has a working electrode with high relative sensitivity because the electrode itself has high conductivity, good sensitivity during measurement, and a low BGC value.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and an object thereof is to exhibit high conductivity, relatively good absolute sensitivity in measurement, and EC with a working electrode that can measure the substance to be measured with relatively high sensitivity due to its low BGC value
To provide D. Another object of the present invention is an EC having a polymer-carbon working electrode having the above-mentioned excellent properties.
To provide D.

(Means and Actions for Solving Problems) The inventors investigated the material of the working electrode in order to develop an ECD capable of measuring a substance to be measured with high sensitivity.
First, regarding the polymer-carbon working electrode, the particle size of graphite carbon was changed, but no significant difference was observed in the performance of the electrode. Next, when the particle shape of the graphite carbon was changed, it was found that the BGC value was lowered when spherical graphite carbon was used, and the present invention was completed.

The electrochemical detector of the present invention is an electrochemical detector for high performance liquid chromatography having a cell having a working electrode, a counter electrode and a reference electrode; and a detection part, wherein the working electrode is a synthetic polymer compound and spherical graphite. It is composed of a composition mainly composed of carbon, whereby the above object is achieved.

The spherical graphite carbon contained in the composition forming the working electrode of the ECD of the present invention has a spherical shape or a shape close to a spherical shape. The particle size is not particularly limited, but is usually 1 to 100 μm. This spherical graphite carbon is 20 to 80% by weight in the composition of the working electrode.
It is contained in the ratio of. If the amount is too small, the conductivity of the working electrode is low and the absolute sensitivity is low. If it is excessive, the BGC value increases and the absolute sensitivity decreases.

The polymer contained in the composition is not particularly limited,
Water that is usually used as an eluent for liquid chromatography; and a material that does not dissolve in or swell with an organic solvent such as methanol or acetonitrile are selected. Examples of such polymers having excellent water resistance and organic solvent resistance include fluororesins, polyphenylene sulfide resins, and furan resins. When the amount of organic solvent in the eluent is small, phenol resin, epoxy resin, etc. can be used.

The working electrode used in the present invention is obtained by mixing the above polymer and spherical graphite carbon, and optionally other additives, and heating and molding. EC of the present invention
As the counter electrode and the reference electrode of D, any of the counter electrode and the reference electrode used in a usual ECD can be used. As the material for the counter electrode, stainless steel (SUS316), which has good conductivity and is resistant to corrosion, is used. A silver / silver chloride electrode or the like is used as the reference electrode. For the detector that measures the current due to the electrochemical reaction of the substance to be measured, a normal ECD detector is used, such as amplifying a minute current and measuring it as a voltage.

ECD cell 1 with working electrode, counter electrode and reference electrode set
The structure of 0 is shown, for example, in the sectional view of FIG.

This ECD cell 10 has cell channels 11 and 12 and a reference electrode 1
5 holds the cell block 1, a block holding plate 3 that rotatably connects the cell block 1 to a cell holder (not shown), and a working electrode 21 attached to the block holding plate 3.
And a working electrode portion 2 for holding

The cell block 1 has a rectangular parallelepiped shape, and has a protrusion 1a at the center of one side surface. The block holding plate 3 has a through hole that fits into the protruding portion 1a, and one side surface of the cell block 1 on which the protruding portion 1a is formed is brought into contact with one side surface of the block holding plate 3 to form the protruding portion. 1a is fitted in the through hole of the block holding plate 3.

The block holding plate 3 has connecting portions 31 and 32 projecting in a direction opposite to the side where the cell block 1 is arranged, on the upper and lower sides of the side surface opposite to the contact surface of the cell block 1, and on the lower side thereof. The connecting portion 32 is pivotally supported by a cell holder (not shown). The upper connecting portion 31 is attachable to and detachable from the cell holder.

At the center of the cell block 1, a pair of upper and lower cell channels 11 and 12 penetrating the cell block 1 in the horizontal direction are arranged. An opening at one end of each of the cell flow paths 11 and 12 is formed on the end surface of the protrusion 1 a of the cell block 1. The cell channel is made of a conductive metal, and this constitutes the liquid contact part 13. A counter electrode (not shown) is provided at the liquid contact portion 13 so as to come into contact with the fluid flowing through the liquid contact portion 13. A reference electrode insertion hole 14 is provided in the upper portion of the cell block 1. The reference electrode insertion hole 14 is opened upward from the upper surface of the cell block 1, and the lower end portion of the reference electrode insertion hole 14 is at the lower end of the cell block 1.
The cell flow path 12 on the upper side of the central portion is reached. A reference electrode is inserted in the reference electrode insertion hole 14.

The working electrode portion 2 is arranged between the connecting portions 31 and 32 of the block holding plate 3. The working electrode portion 2 is fixed to a side surface of the block holding plate 3 opposite to the cell block 1 contact surface by a pair of guide pins 25. The working electrode portion 2 has a working electrode 21 facing the end face of the protrusion 1 a of the cell block 1. The working electrode 21 is supported in a through hole provided in the central portion of a disk-shaped supporting member 22. The support member 22 is supported by the guide pin 25, and the working electrode 21
Are opposed to the open end faces of the cell channels 11 and 12 located in the end face of the protrusion 1a of the cell block 1. The working electrode
A gasket 23 having a through hole in its central portion is interposed between 21 and the end surface of the protrusion 1a of the cell block 1 so that a liquid-tight cell space 16 is formed. And the gasket
The open end faces of the cell channels 11 and 12 are located in the cell space 16 at the center of 23, and the working electrode 21 is
It opposes the open end faces of the cell flow paths 11 and 12 via. The working electrode 21 is pressed by the electrode pressing member 24 supported by the guide pin 25 against the end surface of the protrusion 1a of the cell block 1 via the gasket 23. The electrode pressing member 24 is
An electrode pressing plate 24a made of a relatively soft and highly elastic material in contact with the working electrode 21, an electrode pressing plate 24b made of a material having no elasticity, and an electrode pressing plate 24c made of a high hardness material.

A block cover plate 4 is attached to the side surface of the cell block 1 opposite to the side surface on which the protrusion 1a is formed. The block cover plate 4 has a through hole 4a in the center thereof, and the open end faces of the cell channels 11 and 12 located in the side surface of the cell block 1 are located in the through hole 4a.

When the ECD having the cell 10 is set in high performance liquid chromatography, the effluent containing the substance to be measured from the high performance liquid chromatography passes through the cell flow passage 11, the cell space 16, and the cell flow passage 12 and the cell 10 flows. It is discharged outside. A voltage is applied between the working electrode 21 and the liquid contact part 13, and the effluent containing the substance to be measured undergoes an electrochemical reaction while passing through the cell 10. The current generated at this time is amplified and measured by the detection unit (not shown).

When the substance to be measured is measured using such an ECD of the present invention, the relative sensitivity is extremely excellent. For example, as shown in the following examples, when estriol is measured, the BGC value is about 1/2 as compared with the case of measuring with an ECD having a working electrode using conventional crushed graphite carbon.
That is, it can be seen that the substantially highest detection sensitivity (indicated by the signal peak height / noise level) is about twice that of the conventional one.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Example 1 Epoxy resin Epicoat 828 and curing agent Epicure Z
Spherical graphite with an average particle size of 6 μm (mesocarbon microbeads graphitized product) (manufactured by Osaka Gas Co.) was mixed in a predetermined ratio with a 1: 1 (weight ratio) mixture (all manufactured by Yuka Shell Co., Ltd.). In a closed stainless steel container,
After curing reaction at ℃ for 4 hours, take it out, length 50mm, diameter 12m
A rod-shaped molded body of m was obtained. The obtained compact was sliced to a thickness of 3 mm, and the automatic polishing machine Emmet IV type (BUEHLER LTD.
The surface was polished by A).

The resistance value of the test piece thus obtained was measured. The relationship between the content of spherical graphite carbon in the electrode and the resistance value is shown in Fig. 2 (a). From FIG. 2 (a), it can be seen that the resistance value exceeds 10 ³ Ω when the spherical graphite carbon content falls below 20% by weight. The working electrode must have a certain degree of conductivity, and in order for this test piece to function as a working electrode, it must have a spherical graphite carbon content of at least 20% by weight, preferably about 25% by weight. It can be seen that it is% by weight.

Next, this was attached to the cell shown in FIG. 1 as a working electrode, and this cell was mounted on an ECD-120 type detector (Sekisui Chemical Co., Ltd.).
Set to. This detector was connected to LC6A high performance liquid chromatography (manufactured by Shimadzu Corporation). Epinephrine,
1 μg / norepinephrine and dopamine each
The sample solution contained at a ratio of ml was subjected to the above high performance liquid chromatography for measurement. The measurement conditions are as follows.

Eluent: 0.1 M phosphate buffer (pH 3.5) Flow rate: 1.0 ml / min Applied voltage: 700 mV Sample solution: Epinephrine (E), Norepinephrine, Dopamine (DA) each concentration 1 μg / ml Injection volume: 1 μ Separation Column: Medipora 113N (Sekisui Chemical Co., Ltd.)
The relationship between the content of spherical graphite carbon contained in the working electrode and the peak height (indicating sensitivity) of norepinephrine (NE) at the time of measurement is shown in FIG. 2 (b). From FIG. 2 (b), it can be seen that the sensitivity drops sharply when the spherical graphite carbon content falls below 40% by weight, but a substantially constant sensitivity is obtained above 40% by weight.
From FIG. 2 (b), it is clear that the spherical graphite carbon content must be about 20% by weight or more, preferably about 40% by weight or more.

Furthermore, the content of spherical graphite carbon contained in the working electrode and the background current (BG
The relationship with C.) is shown in Fig. 2 (c). From FIG. 2 (c), it can be seen that BGC rapidly increases when the spherical graphite carbon content exceeds 60% by weight. The spherical graphite carbon content of up to about 90% by weight can be used as an electrode, but it is preferably 80% by weight or less.

It is desirable that the sensitivity of the ECD detector is high, but even if the sensitivity is high, the substantial sensitivity decreases if BGC is high. From FIGS. 2 (a) to (c), it can be seen that spherical graphite carbon of 20 to 80% by weight can be suitably used as a working electrode of ECD because of its low resistance value, good sensitivity, and small BGC. It is clear that the range.

Example 2 Polyphenylene sulfide resin, Ryton GR01 (manufactured by Phillip Oil Co., Ltd.) 40 parts by weight, average particle diameter 6 μm
60 parts by weight of spherical graphite (graphitized mesocarbon microbeads) (manufactured by Osaka Gas Co., Ltd.) was added and mixed gently in a mortar. This is the MINI MAX MOLDAR (COSTOM SCIENTIFI
C INSTRUMENTS Co .; CEDAR KNOLLS NJUSA) was used to mold at a resin temperature of 320 ° C to a thickness of 3 mm and a diameter of 12 mm. The surface of the obtained molded product is automatically polished by Emmet IV type (BUEHLER
Polished by LTD. (USA). The molded body thus obtained was mounted as the working electrode of the cell shown in FIG.
It was set on a CD-120 type detector (manufactured by Sekisui Chemical Co., Ltd.). This detector was connected to LC6A high performance liquid chromatography (Shimadzu). The sample solution containing estritol at a ratio of 4 ng / ml was applied to the above high performance liquid chromatograph for measurement. The measurement conditions are as follows.

Eluent: 50 mM phosphate buffer (pH 3.5) 50% by volume / acetonitrile 35% by volume / methanol 15% by volume Flow rate: 1.0 ml / min Applied voltage: 1000 mV Test substance: Estritol (E ₃ ) Concentration: 4 ng / ml Injection volume : 5μ Separation column: Medipora ODS-113N Sekisui Chemical Co., Ltd. The obtained chromatogram is shown in FIG. The conductive resistance, peak height, and BGC value 60 minutes after power-on are 1 for each.
It was 0.25Ω, 95mm and 47nA. The minimum detection amount (indicating the maximum detection sensitivity) is 2.5 pg (E ₃ peak height / noise level =
It was 3).

Comparative Example 1 The same as Example 2 except that a graphite carbon 325 mesh passing product (manufactured by Tokai Carbon Co., Ltd.) was used in place of the spherical graphite carbon. The conductive resistance value, peak height (sensitivity) and BGC value 60 minutes after power-on are 5.7 Ω, 92.3 mm and 84.7, respectively, and the minimum detection amount is
It was 5.1 pg (E ₃ peak height / noise level = 3). This was about half the sensitivity of Example 2 above.

Example 3 A working electrode was obtained in the same manner as in Example 1, set in the same detector as in Example 1 and subjected to high performance liquid chromatography under the following conditions.

Eluent: 0.1 M phosphate buffer (pH 3.5) 95% by volume / 5% by volume methyl alcohol, 0.5 mM sodium octylsulfonate ECD Applied voltage: 750 mV Test substance: Norepinephrine (NE), Epinephrine (E), 3,4-Hydroxybenzylamine (DHBA), Dopamine (DA) 2pg / μ each Injection volume: 10μ Separation column: Medipora 113N (Sekisui Chemical Co., Ltd.)
Flow rate: 1.0 ml / min. The obtained chromatogram is shown in FIG. From Figure 4,
It can be seen that the sensitivity of each substance to be measured is good even when the sample liquid containing a plurality of substances to be measured is used. The practical minimum NE detection amount is 0.5 pg (NE peak height / noise level = 3)
Met.

(Effects of the Invention) According to the present invention, there is thus provided an electrochemical detector having a working electrode having a specific composition and capable of measuring a substance to be measured with high sensitivity. This detector is advantageously used to measure vitamins, steroids, various drugs, etc. in a sample separated by high performance liquid chromatography.

[Brief description of drawings]

FIG. 1 is a sectional view showing an example of the cell of the ECD of the present invention; FIG. 2 (a) is a relation between the content of spherical graphite carbon in the working electrode of the ECD of the present invention and the resistance value of the electrode. 2B is a graph showing the relationship between the spherical graphite carbon content of the working electrode and the peak of the substance to be measured in the sample; FIG. 2C is the spherical graphite of the working electrode. Graphs showing the relationship between the carbon content and the background current; and FIGS. 3 and 4 are chromatograms when various substances to be measured were measured using the ECD of the present invention. 1 …… Cell block, 10 …… ECD cell, 15 …… Reference electrode, 21
...... Working electrode.

Front page continuation (72) Inventor Hideo Matsumura 5-2, Suzaku, Nara City, Nara Prefecture (72) Hirohito Nishino 536-53, Minamikasacho, Kusatsu City, Shiga Prefecture

Claims (2)

[Claims] 1. An electrochemical detector for high performance liquid chromatography having a cell having a working electrode, a counter electrode and a reference electrode; and a detection part, wherein the working electrode is mainly composed of a synthetic polymer compound and spherical graphite carbon. An electrochemical detector composed of the composition. 2. The composition according to claim 1, which contains the spherical graphite carbon in a proportion of 20 to 80% by weight.
The detector according to item.