JP2005195489A - Liquid chromatograph device and method for analyzing alcohol and saccharide thereby - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a liquid chromatograph device for sensitively detecting alcohol and saccharide for which any suitable detection means has so far been unavailable. <P>SOLUTION: According to this liquid chromatograph analysis method, an eluate from a separation column of a liquid chromatograph is passed through an electrolytic device to electrolyze constituents in a solution and electrolytic current is measured to analyze the constituents. A diamond electrode doped with boron is used as a working electrode of the electrolytic device. Alcohol or saccharide in the solution is detected by measuring electrolytic current with a definite voltage impressed on the working electrode. Since the use of the diamond electrode allows the use of a voltage higher than before for electrolysis, alcohol and saccharide can be analyzed which have not been electrolyzed by low voltages as in the past. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a liquid chromatograph apparatus particularly suitable for analysis of alcohols and saccharides.

  One of the methods for analyzing components in solutions containing various organic compounds is a liquid chromatographic method, and high performance liquid chromatography (HPLC) using a packing with improved particle size distribution in the separation column. Currently popular. This method can also be applied to the analysis of alcohols and saccharides. In this case, a differential refractometer detector is generally used as a means for detecting a component in a solution that has passed through a separation column. However, while this is versatile, there are problems such as lack of selectivity depending on the type of substance and poor sensitivity. On the other hand, an ultraviolet absorption detector is most commonly used as a selective detector, but cannot be applied to alcohols and saccharides because they do not exhibit effective ultraviolet absorption. In addition, there are a fluorescence detector, a chemiluminescence detector, and the like as selective detectors, and the sensitivity is good, but it is difficult to apply to alcohols and saccharides which are the objects of the present invention.

Furthermore, there is an electrochemical detector as shown in JP-A-5-296964 as a detector used in HPLC. This method is used for the analysis of catecholamines and the like, and the above publication also describes the analysis of such physiologically active substances and biological metabolites. The method disclosed in Japanese Patent Application Laid-Open No. 5-296964 uses conventional porous graphite as a coulometer type detector by using glassy carbon having 1-100 μm diameter pores on the surface as an active electrode. It is said that much more sensitive electrochemical conversion characteristics can be obtained.
JP-A-5-296964

  An apparatus combining an HPLC and an electrochemical detector disclosed in Japanese Patent Application Laid-Open No. 5-296964 discloses analysis of catecholamines, serotonin and their metabolites as application examples. And saccharides are not described. In fact, it is impossible to apply this method to detection of alcohols and saccharides. That is, the voltage that can be applied between the electrodes in the electrochemical detector is limited by the occurrence of water electrolysis, but the oxidation reaction of alcohols and sugars does not occur below the voltage at which water electrolysis occurs. The present invention aims to provide a method for detecting alcohols and saccharides with high sensitivity using HPLC due to such problems.

  The present invention solves the above-mentioned problems, and is a liquid chromatograph analyzer provided with an electrolyzer for passing an eluate from a separation column of a liquid chromatograph, wherein the electrolyzer is doped with boron as a working electrode. The liquid chromatograph analyzer is provided with a measuring device configured to measure an electrolytic current value in a state where a constant voltage is applied to the working electrode.

  Furthermore, in the liquid chromatograph analyzer provided with an electrolyzer for passing the eluate from the separation column of the liquid chromatograph, two or more of the electrolyzers are connected in series to the flow path of the eluate. Of these, at least the most downstream electrolyzer is composed of a boron-doped diamond electrode as the working electrode, and each electrolyzer is in a state in which a constant voltage is sequentially applied to the working electrode from the upstream to the downstream electrolyzer. A liquid chromatograph analyzer characterized in that a measuring device for measuring the electrolytic current value is provided.

  In these liquid chromatographic analyzers, an electrolysis apparatus constituted by a boron-doped diamond electrode as a working electrode has a working electrode in which boron-doped diamond is formed on the surface of a conductive substrate; Or a spacer made of an electrical insulator having a thickness of 1 mm and having a hole in the surface, and a counter electrode made of a block of a conductor facing the working electrode across the spacer, and the eluent inlet for the counter electrode The discharge port and the reference electrode are also provided so as to be opened in one space formed by the hole of the spacer.

  The present invention also relates to a liquid chromatograph analysis method for analyzing a component by passing an eluate from a separation column of a liquid chromatograph through an electrolysis device to electrolyze the component in the solution and measuring an electrolysis current. An alcohol characterized in that a boron-doped diamond electrode is used as the working electrode and the alcohol or saccharide in the solution is detected by measuring an electrolytic current value with a constant voltage applied to the working electrode. This is a method for analyzing saccharides or sugars.

  Since the liquid chromatograph apparatus of the present invention can be electrolyzed by applying a higher voltage than before in an electrolysis apparatus that detects components in the effluent from the separation column, the range of compounds to be detected is conventionally limited. It can be expanded to those not electrolyzed at a low voltage. In particular, when the liquid chromatograph apparatus of the present invention is applied to the detection of alcohols and saccharides for which there has been no suitable method capable of detecting with high sensitivity in the conventional liquid chromatographic analysis, an excellent effect can be exhibited.

  FIG. 1 is a conceptual diagram showing the configuration of the liquid chromatograph analyzer of the present invention. The eluent 1 is supplied to the separation column 5 at a constant speed by a pump 3 through a degasser 2 that removes dissolved gas by decompression. Reference numeral 4 denotes an injector, which injects a predetermined amount of a test sample into the separation column 5. The eluate discharged from the separation column 5 is sent to the electrolysis device 7 and is detected by the measurement device 8 every time each electrochemically active component is eluted from the separation column. In the electrolyzer 7, a constant voltage is applied between the electrodes, and detection is performed by detecting an electrolysis current due to the solution component.

The separation column 6 preferably has a large difference in adsorption power with respect to each component to be analyzed. For the analysis of alcohols and saccharides that the liquid chromatograph analyzer of the present invention is particularly intended for, for example, alcohol Polymeric ion exchange columns can be used for types, and normal phase amino group-bonded columns can be used for saccharides. As an eluent for this, for example, an acetonitrile solution in which KH ₂ PO ₄ is added as an electrolyte for electrolysis can be used.

  2A and 2B are diagrams showing an electrolysis apparatus in the liquid chromatograph apparatus of the present invention, wherein FIG. 2A is a sectional view and FIG. 2B is a front view with a part removed. In FIG. 2, 11 is a working electrode of the electrolysis apparatus, and a conductive diamond film is formed on the surface (the upper surface in FIG. 2A) on the side where the eluate from the column of the conductive silicon plate contacts. Has been. This working electrode is provided with a metal block 12 for reinforcement, and a lead wire 13 is coupled thereto.

  A counter electrode 14 is made of a conductive block having corrosion resistance such as titanium and sandwiches a spacer 15 made of a sheet of an electrical insulator such as fluorine resin having a thickness of 0.1 to 1 mm. It faces the working electrode 11. FIG. 2B is a view corresponding to a cross section taken along the line AA ′ in FIG. 2A with the working electrode 11 removed. As shown in FIG. 2B, the spacer 15 has one elongated hole 16. Is open. As shown in FIG. 2A, the hole 16 forms a thin plate-like space 17 sandwiched between the working electrode 11 and the counter electrode 14 in the assembled state of the electrolysis apparatus, and is removed from the column. It becomes the flow path of the eluate.

  Further, the counter electrode 14 is provided with an inlet port 18 and an outlet port 19 for the eluate and a reference electrode 20 which are opened in one space 17 formed by the hole 16 of the spacer 15. It is convenient that at least one of the joints 21 and 22 of the introduction port 18 and the discharge port 19 is made of metal, and any one of the pipes 23 and 24 made of stainless steel or the like is used as an energization circuit to the counter electrode 14. The reference electrode 20 is an Ag-AgCl-based example. However, a chemical-resistant container 25 such as a fluororesin in which a KCl solution is gelatinized with gelatin is filled as an electrolyte solution 26. . Further, an Ag wire having a surface made of AgCl is inserted as an electrode material 27 therein. Reference numeral 28 denotes a filter made of porous ceramics that separates the reference electrode from the liquid to be measured. In the present invention, the reference electrode is not limited to the Ag-AgCl system, and other compositions can be used.

  As described above, a conductive diamond electrode is used in the electrolysis apparatus according to the present invention. However, conductivity can be imparted by doping diamond with boron, and such diamond is deposited on the conductive substrate. And a thickness of about 30 μm. In order to form conductive diamond, a conductive silicon plate is used as a substrate, and the surface thereof is polished and accommodated in a reaction vessel of a plasma CVD apparatus, and is brought into a reduced pressure state of about 100 Torr and contains a carbon source. A microwave discharge is generated in a hydrogen gas atmosphere. A mixture of acetone and methanol is used as the carbon source, and a small amount of boron oxide (for example, 1% in the ratio of boron / carbon) is dissolved in order to dope boron. The temperature of the substrate reaches about 900 ° C. by the plasma discharge, and a film made of fine diamond crystals with a size of several μm is gradually formed.

  As described above, in the electrolysis apparatus of the present invention, the working electrode and the counter electrode are configured such that the surfaces with a narrow interval face each other. Thus, by making the working electrode and the counter electrode completely face each other, the entire surface of the diamond electrode as the working electrode can be effectively contributed to the electrolytic action, and the rate of the electrolytic reaction can be sufficiently increased. In addition, by narrowing the distance between the working electrode and the counter electrode, the eluate from the column can quickly pass through, and the tailing phenomenon can occur in which the previous eluate stays and the peak of the measured value falls. No. Accordingly, the resolution of the chromatograph can be increased in combination with the high rate of the electrolytic reaction as described above. The distance between the working electrode and the counter electrode is determined by the thickness of the spacer 15 as described above, but 0.1 to 1 mm is appropriate. If the interval is smaller than 0.1 mm, the resistance of the liquid flow becomes too large, and if it is larger than 1 mm, the amount of mixture of the eluate before and after becomes large and the resolution of the chromatograph is lowered.

  In the electrolysis apparatus used as a detector in the chromatographic apparatus disclosed in JP-A-5-296964 described as the prior art, an example in which glassy carbon is used as a working electrode is shown. In this conventional electrolyzer using glassy carbon, including this example, cylindrical glassy carbon is placed in a container of an electrical insulator corresponding to this, and an external lead wire is connected to glassy carbon. The working electrode is used. In addition, the electrical insulator container is connected to pipes for feeding and discharging the liquid to be electrolyzed. The pipe is made of metal and energized to form a counter electrode. The structure of the electrolyzer according to the present invention is completely different from that of the conventional electrolyzer as described above, but unlike the glassy carbon having a porous and three-dimensional reaction region, the performance of the diamond electrode having a flat reaction region is improved. As a result of various experiments to achieve the maximum, the structure as in the present invention has been reached.

  In the above-described electrolysis apparatus, the eluate from the separation column is continuously fed in a state where constant electrolysis conditions are set, and the components contained in the liquid are sequentially detected. The most common electrolysis condition is that a constant voltage is applied between the working electrode and the counter electrode, and the change in the electrolysis current during that time is measured and recorded, so that the eluate from the separation column contains components. Are detected sequentially.

  By the way, in order for electrolysis to occur, it is necessary to apply more than the electrolysis voltage peculiar to each component between the electrodes. In the case of amines, the electrolysis voltage is relatively low, but in the case of alcohols and saccharides, the electrolysis voltage is high, so that it cannot be measured by a conventional electrolysis apparatus as described above. That is, in an electrolysis apparatus conventionally used as a detector for the eluate component of a liquid chromatograph, a carbonaceous material such as glassy carbon has been used as a working electrode. However, in the case of a carbonaceous working electrode, electrolysis of water becomes intense when the voltage exceeds about 1.2 V in relation to hydrogen overvoltage, and measurement with a voltage higher than this becomes impossible. However, in the electrolysis apparatus using the diamond electrode according to the present invention, since the hydrogen overvoltage is high, electrolysis of water does not occur up to about 2.5 V, and an electrolysis voltage exceeding 1.2 V can be applied.

  As described above, since the voltage of the electrolysis apparatus can be made higher than before, alcohols such as methanol, ethanol, propanol, ethylene glycol, glycerin, ribose, glucose, Analysis of sugars such as lactose, fructose, inositol and sorbitol is now possible. Therefore, these components can be separated and detected in a solution containing one or both of alcohols and saccharides.

  In addition, when a high voltage capable of electrolyzing alcohols and saccharides is applied, amines having a low electrolysis voltage will be detected, but another electrolyzer with a low electrolysis voltage is placed on the front side of the flow path. If it is provided and electrolyzed with amines or the like before detection, it can be separated from these. In this way, two or more electrolyzers are connected in series to the flow path of the eluate, and in each electrolyzer from upstream to downstream, each constant high voltage is applied to the working electrode in sequence. By measuring the electrolysis current value, it is possible to classify alcohols and saccharides by electrolysis voltage to further easily identify the components. In this case, if the purpose is only separation from amines having a relatively low electrolysis voltage, it is not necessary to use a boron-doped diamond electrode as the working electrode in the previous electrolysis apparatus, and electrolysis of water must occur. For example, a carbonaceous working electrode may be used. Eventually, the object of the present invention of analyzing alcohols and saccharides can be achieved if at least the most downstream of the two or more electrolyzers is composed of a boron-doped diamond electrode as the working electrode.

Analysis was performed using the apparatus shown in FIG. 100 mMol-KH ₂ as eluent 1
PO ₄ + 10% acetonitrile aqueous solution was supplied at a rate of 0.5 ml / min. As the column 4, an ion exchange column having an inner diameter of 4.6 mm and a length of 250 mm was used. The electrolyzer 7 has the structure shown in FIG. 2 and uses diamond doped with boron as a working electrode. The electrolyzer is connected to a coulometer type measuring device 8 that records a current value measured with a constant voltage applied to the working electrode over time.

  The working electrode applied voltage of the electrolyzer was set to 2.5 V (relative to the Ag / AgCl reference electrode), but water electrolysis did not occur. When samples of methanol, ethanol, and 2-propanol each having a concentration of 1 pmol / μl were injected from the 20 μl injector 4, the peak of the electrolysis current of each component was subsequently detected. On the other hand, when the electrolysis apparatus is changed to one using the conventional glassy carbon electrode instead of the above diamond electrode, and 1.15 V (applied to the reference electrode) is applied without electrolysis of water. The peak of the electrolysis current was not detected even when the sample solution was injected into the injector.

  Also, column 4 of the above conditions was changed to a normal-phase amino group-binding column, and the other conditions were the same under the same conditions: ribose, glucose, and lactose were injected from 20 μl injector 4 at a concentration of 5 pmol / μl each. Thereafter, the peak of the electrolysis current of each component was detected. On the other hand, when the electrolysis apparatus is changed to one using a glassy carbon electrode instead of a diamond electrode, and 1.15 V (applicable to the reference electrode) is applied without electrolysis of water, Even when the sample solution was injected, the peak of the electrolysis current was not detected.

The conceptual diagram which shows the structure of the liquid chromatograph analyzer of this invention It is a figure which shows the electrolysis apparatus in this invention, Comprising: (a) is sectional drawing, (b) is the front view which removed a part

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Eluent 2 Degasser 3 Pump 4 Injector 5 Separation column 7 Electrolyzer 8 Measuring device 11 Working electrode 12 Metal block 13 Lead wire 14 Counter electrode 15 Spacer 16 Hole 17 Space 18 Eluent inlet 19 Elutant outlet 20 Reference electrode 21, 22 Joint 23, 24 Piping 25 Container 26 Electrolyte 27 Electrode material 28 Filter

Claims (4)

In a liquid chromatographic analyzer provided with an electrolyzer for passing an eluate from a separation column of a liquid chromatograph, the electrolyzer is constituted by a diamond electrode doped with boron as a working electrode, and the working electrode has a fixed amount. A liquid chromatograph analyzer characterized in that a measuring device for measuring an electrolysis current value in a state where a voltage is applied is provided. In the liquid chromatograph analyzer provided with an electrolyzer for passing the eluate from the separation column of the liquid chromatograph, two or more electrolyzers are connected in series to the flow path of the eluate, at least of which The most downstream electrolyzer is composed of a boron-doped diamond electrode as a working electrode, and the electrolysis current of each electrolyzer is applied to each working electrode in a state where a high voltage is applied to the working electrode in order from the upstream to the downstream electrolyzer. A liquid chromatograph analyzer characterized in that a measuring device for measuring a value is provided. An electrolysis apparatus constituted by a boron-doped diamond electrode as a working electrode has a working electrode in which boron-doped diamond is formed on the surface of a conductive substrate, and a hole in the surface with a thickness of 0.1 to 1 mm. A spacer made of an electrical insulator having an opening and a counter electrode made of a conductive block facing the working electrode across the spacer, and the counter electrode has an eluate inlet and outlet and a reference electrode, 3. The liquid chromatograph analyzer according to claim 1, wherein the liquid chromatograph analyzer is provided so as to open in one space formed by a hole of the spacer. In a liquid chromatographic analysis method in which an eluate from a separation column of a liquid chromatograph is passed through an electrolysis apparatus to electrolyze the components in the solution and an electrolytic current is measured, boron is used as a working electrode of the electrolysis apparatus. Analysis of alcohols or saccharides, characterized in that an alcohol or saccharide in the solution is detected by measuring an electrolysis current value with a constant voltage applied to the working electrode using a diamond electrode doped with Method.

Images