JP5116388B2 - Method and apparatus for evaluating optical isomer resolution of chiral stationary phase for chromatography - Google Patents

Description

  The present invention relates to an evaluation method and an evaluation apparatus for optical isomer resolution of a chiral stationary phase for chromatography suitable for separation of optical isomers in pharmaceuticals and the like.

Although the general physicochemical properties are exactly the same among optical isomers, the behavior and physiological activity in vivo may differ. Therefore, in the development of pharmaceuticals that are specific optical isomers, for example, research on the purity, pharmacology, and toxicological evaluation of synthetic products is necessary. In addition, “chiral separation” in which a specific optical isomer is detected separately from other optical isomers is a very important issue. As a useful method for performing chiral separation, a technique using high performance liquid chromatography (hereinafter abbreviated as HPLC) is exemplified (for example, refer to Patent Document 1). At present, optical isomers such as pharmaceuticals and biological samples are used. A large number of chiral stationary phases have been developed to separate the chiral columns, and chiral columns equipped with such chiral stationary phases are widely used. Here, the term “chiral stationary phase” refers to all substances for separating optical isomers that are bound to a carrier of a chiral column.
Japanese Patent Laid-Open No. 11-23552

  However, in practice, when performing chiral separation by HPLC of the target sample optical isomers, it was necessary to select a chiral stationary phase based on previous experience, and it was necessary to repeat trial and error. . In other words, it is necessary to purchase many kinds of chiral columns that are more expensive than ordinary HPLC columns, and to optimize the analysis conditions such as the composition and flow rate of the mobile phase, and these operations require a great amount of labor, time and cost. It was a thing. Therefore, a technique for performing these operations efficiently and at low cost is very useful, and its development is strongly demanded.

  The present invention has been made in view of the above circumstances, and it is an object of the present invention to provide a rapid, simple and low-cost method for evaluating the resolution of an optical isomer of a chiral stationary phase for chromatography and an evaluation apparatus used therefor. .

  As a result of diligent research, the inventors of the present invention bonded a compound that can be used as a chiral stationary phase on a gold electrode of a quartz resonator microbalance (QCM) sensor to form a pseudo chiral stationary phase, After stabilizing this in the mobile phase solution, a sample containing a specific target optical isomer was added, and the frequency variation of the sensor was observed. Based on the interaction with the chiral stationary phase The optical isomers can be detected individually using the frequency change as an index, and the elution order of the optical isomers from the chiral column can be determined based on the information at this time, and the resolution value at that time can also be estimated. As a result, the present invention has been completed.

That is, to solve the above problem,
The invention according to claim 1 is a method for evaluating the optical isomer separation ability of a chiral stationary phase for separating and detecting optical isomers to be detected, which is applied to one or both electrodes of a piezoelectric element. An agent for chiral stationary phase, which interacts with the optical isomer to be detected, is immobilized, and is linked to the electrode at one end and a linking substance having a linear chain with a carboxyl group at the other end. The agent bound to avidin and having an amino group is bound to biotin and further forms a biotin-avidin complex, whereby the agent is immobilized on the electrode via a non-covalent bond, by formation and eliminating non-covalent, wherein the agent is a can be removed from the fixed and the electrode to the electrode, the electrode, the circuit and the frequency measuring device for applying a voltage between the electrodes In a solution containing an optical isomer to be detected in the solution tank, using an evaluation cell comprising a sensor part capable of being electrically connected and a solution tank in which the sensor part is immersed in a liquid , While applying a voltage between the electrodes of the sensor unit to vibrate the piezoelectric element, the frequency variation of the piezoelectric element due to the interaction between the optical isomer to be detected and the active substance is measured, and further, instead of the solution, This is a method for evaluating optical isomer resolution, comprising measuring frequency fluctuations using a solution containing optical isomers not to be detected and comparing the obtained measurement results.
The invention according to claim 2 is a method for evaluating the optical isomer separation ability of a chiral stationary phase for separating and detecting optical isomers to be detected, which is applied to one or both electrodes of a piezoelectric element. An agent for chiral stationary phase, which interacts with the optical isomer to be detected, is immobilized, and is linked to the electrode at one end, and a linking substance having a linear main chain having an amino group at the other end. The active substance bound to biotin and having a carboxyl group is bound to avidin and further forms a biotin-avidin complex, so that the active substance is fixed to the electrode via a non-covalent bond, The active substance can be fixed to and removed from the electrodes by forming and eliminating non-covalent bonds, and the electrode applies a voltage between the electrodes and a frequency measuring device. In a solution containing an optical isomer to be detected in the solution tank, using an evaluation cell comprising a sensor part capable of being electrically connected and a solution tank in which the sensor part is immersed in a liquid , While applying a voltage between the electrodes of the sensor unit to vibrate the piezoelectric element, the frequency variation of the piezoelectric element due to the interaction between the optical isomer to be detected and the active substance is measured, and further, instead of the solution, This is a method for evaluating optical isomer resolution, comprising measuring frequency fluctuations using a solution containing optical isomers not to be detected and comparing the obtained measurement results.
According to a third aspect of the present invention, the electrode is a gold electrode, the connecting substance is a linear carboxyalkanethiol, carboxyalkenethiol, or carboxyalkynethiol, and the connecting substance has a sulfur atom in the gold electrode. The method for evaluating optical isomer resolution according to claim 1, wherein the optical isomer separation ability is bonded to a surface .
In the invention according to claim 4, the electrode is a gold electrode, the connecting substance is a linear aminoalkanethiol, aminoalkenethiol, or aminoalkynethiol, and the connecting substance has a sulfur atom in the gold electrode. 3. The method for evaluating optical isomer separation ability according to claim 2, wherein the optical isomer separation ability is bound to a surface .
The invention according to claim 5 is the method for evaluating optical isomer resolution according to any one of claims 1 to 4 , wherein the piezoelectric element is a crystal resonator .
The invention according to claim 6 is the method for evaluating optical isomer resolution according to claim 4 or 5, wherein the aminoalkanethiol has 1 to 4 carbon atoms.
Invention of Claim 7 is an evaluation apparatus used for the evaluation method of the optical isomer separation ability as described in any one of Claims 1 and 3-6, Comprising: On one or both electrodes of a piezoelectric element, An evaluation cell comprising a sensor unit that has an active substance for chiral stationary phase that interacts with an optical isomer to be detected, and a solution bath that immerses the sensor unit in a liquid. The cell includes a plurality of types of sensor units having different active substances, and the electrodes are electrically connected to a circuit and a frequency measuring device for applying a voltage between the electrodes. A solution supply means for supplying a solution, wherein the main chain having a carboxyl group at one end and fixed to the electrode at the other end is bonded to avidin and the amino group has an amino group. Substance binds to biotin Further, by forming a biotin-avidin complex, the active substance is fixed to the electrode through a non-covalent bond, and the active substance is fixed to the electrode by forming and eliminating the non-covalent bond. And an apparatus for evaluating the separation ability of optical isomers, characterized in that it can be removed from the electrode.
Invention of Claim 8 is an evaluation apparatus used for the evaluation method of the optical isomer separation ability as described in any one of Claims 2-6, Comprising: It is a detection object to one or both electrodes of a piezoelectric element. Comprising an evaluation cell comprising a sensor part in which an agent for chiral stationary phase that interacts with the optical isomer of is fixed, and a solution tank in which the sensor part is immersed in a liquid, A plurality of types of sensor units having different active substances are provided, the electrodes are electrically connected to a circuit and a frequency measuring device for applying a voltage between the electrodes, and a solution is supplied to the evaluation cell. A solution supply means for supplying, the main chain having an amino group at the other end fixed to the electrode, and a main chain having an amino group bonded to biotin, and the active substance having a carboxyl group Combined with avidin Further, by forming a biotin-avidin complex, the active substance is fixed to the electrode through a non-covalent bond, and the active substance is fixed to the electrode and the electrode by forming and eliminating the non-covalent bond. It is an apparatus for evaluating the ability to separate optical isomers, which can be removed from water.

  According to the present invention, a rapid and simple screening of a chiral stationary phase suitable for chiral separation of a target sample is possible. In addition, it is not necessary to prepare a large number of different chiral columns, the amount of the active substance for the chiral stationary phase to be used is very small, and the piezoelectric element can be repeatedly used, so that screening can be performed at low cost.

The present invention will be described in detail below.
Piezoelectric elements are obtained by providing a metal thin film on both sides of a slice obtained by cutting a piezoelectric crystal into a very thin plate, and a crystal resonator using a crystal of crystal is known.
When a voltage is applied to the metal thin film of the piezoelectric element, the piezoelectric element vibrates at a certain frequency (resonance frequency). Even if a very small amount of substance of about several ng is adsorbed on the metal thin film, the resonance frequency decreases according to its mass, so that it can be used as a microbalance.
In the present invention, by constructing a chiral stationary phase on the electrode of this piezoelectric element, a pseudo chiral column is prepared, and the difference in the interaction between the optical isomers of the target chiral molecule is measured. Evaluate the optical isomer resolution of the stationary phase.

<Evaluation cell>
FIG. 1 is a diagram illustrating a measurement cell used in the present invention, (a) is a perspective view of a state in which a sensor unit is immersed in a solution in a solution tank, (b) is a front view, and (c) ) Is a cross-sectional view taken along the line AA.
The evaluation cell 7 includes a sensor unit 5 using a crystal resonator as a sensor, and a solution tank 6 for immersing the sensor unit 5 in a solution containing an optical isomer to be measured.
The sensor unit 5 is held by a holding means (not shown) capable of adjusting the arrangement position in the solution tank 6 and can be arranged in the solution tank 6 at the time of measurement and outside the solution tank 6 at times other than the measurement. It is said that.

  Reference numeral 2 denotes a quartz plate, which is sandwiched between a pair of electrodes 3 composed of a first electrode 3a and a second electrode 3b, thereby constituting a quartz crystal resonator 1. One end of the first connection electrode 31a is connected to the first electrode 3a, and one end of the second connection electrode 31b is connected to the second electrode 3b. Further, the quartz plate 2 is exposed so that the plane on the first electrode 3a side is exposed to the outside, and the first electrode 3a is exposed so that the surface facing the contact surface with the quartz plate 2 is exposed to the outside. It is housed in a resin cartridge 4. The first connection electrode 31a and the second connection electrode 31b are housed in the cartridge 4 so that the other ends 311a and 311b are exposed to the outside. That is, the quartz plate 2, the first electrode 3a, the second electrode 3b, the first connection electrode 31a, and the second electrode 3b are accommodated in the cartridge 4, and the sensor unit 5 is formed. The sensor unit 5 is detachable in a detection device (not shown). When the sensor unit 5 is mounted, the other end 311a of the first connection electrode 31a and the other end 311b of the second connection electrode 31b are respectively It is electrically connected to a circuit (not shown) for applying a voltage between the first electrode 3a and the second electrode 3b. Further, the circuit is electrically connected to a frequency measuring device (not shown) for detecting the frequency of vibration of the crystal resonator 1 when a voltage is applied between the first electrode 3a and the second electrode 3b. ing. With this configuration, the crystal unit 1 can be easily replaced by replacing the sensor unit 5.

  Here, the crystal resonator 1 or the like is shown contained in the cartridge 4, but it is not necessarily required to be contained in the cartridge. Further, as shown here, the first electrode 3a and the second electrode 3b are not connected to the first connection electrode 31a and the second connection electrode 31b, and a voltage is applied between the first electrode 3a and the second electrode 3b. You may connect directly to this circuit via wiring.

  The first electrode 3a and the second electrode 3b are plate-shaped, and one surface thereof is provided in contact with the quartz plate 2. And the active substance for chiral stationary phases is being fixed to the surface facing the contact surface with the quartz plate 2 of the 1st electrode 3a.

An agent is a specific optical isomer that can be used for a chiral stationary phase of a chiral column and interacts with an optical isomer to be detected, and is usually different from a measurement target. Here, the interaction means that a bond is formed by an intermolecular force between the optical isomer to be detected and the agent, and specifically, for example, electrostatic interaction, hydrogen bond, hydrophobic bond It means that a bond is formed between molecules due to dipole interaction or van der Waals force.
In general, the stronger the interaction between an active substance and an optical isomer, the higher the retention of the optical isomer in a chiral column using the active substance as a chiral stationary phase.

What is necessary is just to select the said active substance suitably according to the kind of optical isomer. At that time, the active substance can also be selected with reference to the kind of chiral stationary phase of a commercially available chiral column.
Moreover, it is preferable that the kind of active substance to fix is one type per 1st electrode 3a. If a plurality of types of active substances are fixed to each first electrode 3a, the evaluation accuracy may be lowered.

  The optical isomer is a molecule, and may be selected as desired. Regardless of whether it is natural or non-natural, it may be either a low molecule or a polymer, and may be either an inorganic compound or an organic compound, or a complex thereof. . Furthermore, a metal atom may be included. Among them, compounds having physiological activity are suitable, and various organic compounds in which a large number of optical isomers exist as chiral molecules and their chiral separation is usually very difficult are particularly suitable. Specific examples include proteins, peptides, amino acids, DNA, RNA, polysaccharides, oligosaccharides, monosaccharides, lipids, aromatic compounds, heterocyclic compounds, complexes thereof, and those obtained by chemically modifying them.

  The method for fixing the active substance to the first electrode 3a is not particularly limited as long as it can be stably fixed. For example, the active substance may be fixed through a covalent bond, hydrogen bonding, etc. It may be fixed through a non-covalent bond. Furthermore, a connecting substance may be interposed between the active substance and the first electrode 3a.

For example, the following method can be exemplified as a method of immobilizing an agent having an amino group to an electrode via a covalent bond.
That is, as a linking substance on the fixed surface of the electrode, the main chain has a functional group that easily reacts with a metal, preferably at one end of the molecule, and further has a carboxyl group, preferably at the other end of the molecule. The chain-like first organic compound is fixed, and the carboxyl group is activated by, for example, N-hydroxysuccinimide and 1-ethyl-3- (dimethylaminopropyl) carbodiimide, and the activated carboxyl group is bonded to the active substance. A method for bonding an amino group can be exemplified (hereinafter abbreviated as method (1)).

  When the electrode is a gold electrode, the first organic compound is preferably a linear thiol compound having a mercapto group at one end of the molecule and a carboxyl group at the other end of the molecule, Specific examples include linear carboxyalkanethiol, carboxyalkenethiol, or carboxyalkynethiol. Among these, those having 1 to 4 carbon atoms are preferable, and 3-mercaptopropionic acid is particularly preferable. For example, when 3-mercaptopropionic acid is used as the linking substance, 3,3′-dithiodipropionic acid is preferably reacted with a gold electrode.

  As a method of immobilizing an agent having an amino group to the electrode via a non-covalent bond, biotin is directly or indirectly bound to either the electrode fixing surface or the agent, and avidin is bound to the other. -The method of fixing an active substance to an electrode can be illustrated by forming an avidin complex. For example, when avidin is bound to the fixed surface of the electrode, avidin may be used in place of the agent having an amino group in the above method (1). In this case, the agent having an amino group is conventionally used. What is necessary is just to couple | bond biotin with a well-known method.

On the other hand, the following method can be exemplified as a method for fixing an active substance having a carboxyl group to an electrode via a covalent bond.
That is, as a linking substance on the fixed surface of the electrode, the main chain has a functional group that easily reacts with a metal, preferably at one end of the molecule, and an amino group, preferably at the other end of the molecule. The chain-like second organic compound is fixed, the carboxyl group of the active substance is activated in the same manner as in the above method (1), and the amino group of the second organic compound is added to the activated carboxyl group. Examples of the method for bonding the group can be given.

  When the electrode is a gold electrode, the second organic compound is preferably a linear thiol compound having a mercapto group at one end of the molecule and an amino group at the other end of the molecule, Specific examples include linear aminoalkanethiols, aminoalkenethiols, and aminoalkynethiols, with linear aminoalkanethiols being more preferred. The aminoalkanethiol preferably has 1 to 4 carbon atoms, and aminoethanethiol is particularly preferable.

  Examples of the method for immobilizing an agent having a carboxyl group to an electrode through a non-covalent bond include the same methods as in the case of the aforementioned agent having an amino group. For example, avidin is bonded to the immobilization surface of the electrode. Then, biotin may be bound to an active substance having a carboxyl group by a conventionally known method to form a biotin-avidin complex.

  When the thiol compound is used as the first or second organic compound, sulfur atoms in the thiol compound are bonded to gold, and a self-assembled monolayer (hereinafter referred to as SAM) is formed on the gold electrode surface. For short). Since the gold-sulfur bond is relatively stable, the SAM formed is also stable. And since the carboxyl group or amino group at the molecular end of the thiol compound is oriented on the gold electrode surface with the sulfur atom in between, the reaction with the amino group or activated carboxyl group of the active substance can be easily performed. it can.

  Various reactions performed when the active substance is fixed to the first electrode 3a include, for example, fixing a solution containing a raw material used for the reaction such as the thiol compound or 3,3′-dithiodipropionic acid to the first electrode 3a. What is necessary is just to carry out by dripping on the surface or immersing the fixed surface of the 1st electrode 3a in the solution containing the raw material used for reaction.

The optical isomer separation ability can be usually evaluated with higher accuracy as the distance from the electrode fixing surface to the terminal that is not involved in the fixation of the active substance to the electrode becomes uniform. For this purpose, it is necessary to remove foreign substances from the fixed surface of the electrode and to flatten the fixed surface.
In this way, in order to flatten the fixed surface of the electrode, for example, the fixed surface may be treated with an acid.

The acid used for the treatment is preferably a strong acid. For example, hydrochloric acid, sulfuric acid, nitric acid, hydrofluoric acid, 30% hydrogen peroxide solution / concentrated sulfuric acid = 1/3 (v / v) (hereinafter abbreviated as a piranha solution) Etc. Of these, a piranha solution is more preferred because of its high treatment effect.
Examples of the method for treating the fixed surface of the electrode with these acids include a method in which an acid is mounted on the fixed surface and preferably allowed to stand for 2 to 10 minutes and then washed with distilled water twice. .
One type of acid may be used for the treatment, or the treatment may be repeated sequentially using different types of acids.

The amount of the active substance fixed to the first electrode 3a is not particularly limited as long as the effect of the present invention is not hindered.
And the fixed amount can be adjusted by adjusting the fixed amount to the 1st electrode 3a of the connection substance which connects an active substance and the 1st electrode 3a, such as the said thiol compound. And the fixed amount to the 1st electrode 3a of the said connection substance should just adjust the usage-amount of the said connection substance with which it reacts with the 1st electrode 3a.

  Various reactions performed when the active substance is fixed to the first electrode 3a are performed by applying a voltage between the first electrode 3a and the second electrode 3b to vibrate the crystal unit 1 and observing the frequency variation thereof. It is good to do it while observing the degree of.

  The active substance may be fixed to both the first electrode 3a and the second electrode 3b or only to the second electrode 3b, for example, when the crystal resonator 1 or the like is not accommodated in the cartridge 4. May be.

  The material of the first electrode 3a and the second electrode 3b may be any conductive material, but is preferably a corrosion-resistant metal such as gold or platinum, is highly versatile, and can easily fix the active substance as described above. For this reason, gold is particularly preferable.

As the crystal unit 1, a conventionally known one can be used. However, since the frequency when the crystal unit 1 vibrates is determined by the shape and size of the crystal plate 2, the frequency required according to the purpose is set. The shape and size of the quartz plate 2 may be determined in consideration.
When detecting the interaction between the active substance and the optical isomer, the frequency at the time of vibration of the crystal unit 1 is preferably 5 to 40 MHz. Considering sensitivity and handling of the crystal, it is 27 to 40 MHz. More preferably.
The crystal plate 2, the first electrode 3a, and the second electrode 3b preferably have such sizes and shapes that enable such vibration. Specifically, the crystal plate 2 is preferably a disc having a diameter of 3 to 15 mm and a thickness of 50 to 200 μm, and more preferably a disc having a diameter of 7 to 10 mm and a thickness of 50 to 70 μm. . The first electrode 3a and the second electrode 3b are preferably in the form of a disk having a diameter of 1 to 5 mm and a thickness of 150 to 300 nm, more preferably a disk having a diameter of 1 to 3 mm and a thickness of 240 to 280 nm. preferable.
In addition, although the disk-shaped thing is illustrated here as a crystal oscillator, shape is not limited to this, As long as it is plate shape, the shape of the radial direction cross section may be any polygonal shape.

The material of the solution tank 6 may be any material as long as it can hold the solution to be filled and does not change in use, and may be appropriately selected according to the type of the solution containing the optical isomer to be measured. Especially, what has insulation is preferable, and what has insulation and is transparent is more preferable. By having an insulating property, even a small amount of optical isomer can be measured with higher accuracy. Further, since it is transparent, optical analysis such as measurement of absorbance, chemiluminescence or fluorescence emission of the filled solution can be performed in combination, and more detailed measurement is possible. When optical analysis is performed, a commonly used analysis means such as a spectrophotometer may be disposed in the vicinity of the dissolution tank 6.
From the above viewpoint, colorless glass is most preferable as the material of the solution tank 6.

The shape of the solution layer 6 may be any shape as long as it can hold the solution to be filled. Here, an ordinary container-type solution layer in the form of storing a solution is shown, but besides this, for example, an inflow port for allowing the solution to flow in and an exhaust port for discharging the solution are provided separately, It may be a flow cell type that constitutes a part of the circulation path of the solution.
Further, the volume of the solution tank 6 may be appropriately adjusted according to the size of the crystal resonator 1, but is usually preferably 0.5 to 20 mL, and more preferably 0.5 to 11 mL.

  The arrangement direction of the sensor unit 5 in the solution tank 6 is not particularly limited, and as shown in FIG. 1, the active substance fixing surface of the first electrode 3 a may be perpendicular to or parallel to the liquid surface. It does not have to be any of these, and it may be appropriately selected according to the purpose. For example, if the active substance fixing surface of the first electrode 3a is perpendicular to the liquid surface, it is preferable in that the liquid can be easily removed when the crystal unit 1 is cleaned, and workability is improved.

  Here, an example in which one sensor unit 5 is provided in the evaluation cell 7 is shown, but a plurality of sensor units 5 may be provided. In the case where a plurality of sensor units 5 are provided, the evaluation efficiency of the optical isomer resolution can be increased as the number increases as described later. On the other hand, the smaller the number, the lower the manufacturing cost. Considering the balance of these effects, it is preferable that the number of sensor units 5 provided in one solution tank 6 is two to five.

  When a plurality of sensor units 5 are provided, the active substance fixed to the first electrode 3a may be different for each sensor unit 5 or the same, but it is preferable that they are different. By providing a plurality of types of sensor units 5 in which different substances are fixed to the first electrode 3a in this way, as will be described later, the optical isomer separation ability can be evaluated very efficiently.

  When a plurality of sensor units 5 are provided, the arrangement form is not particularly limited, and may be appropriately selected in consideration of the shape and size of the solution tank 6.

  The plurality of sensor units 5 may be (I) arranged so as to face each other, (II) may be arranged in directions orthogonal to each other, or (III) the first electrodes 3a are substantially formed. You may arrange | position so that it may exist in the same surface. Furthermore, none of these may be necessary. Among these, when arranged as in (III), for example, optical analysis such as absorbance, chemiluminescence or fluorescence emission of the solution in the vicinity of the first electrode 3a of each sensor unit 5 can be easily performed. This is preferable.

  Although an example in which a crystal resonator is used as the piezoelectric element is shown here, the present invention is not limited to this. For example, an APM (Acoustic Plate Mode Sensor) device, an FPW (Flexible Plate-Wave Sensor) device, and a SAW device A (Surface Acoustic-Wave Sensor) device or the like can also be used. That is, any plate-like piezoelectric body that resonates at a specific frequency when an external voltage is applied can be used in place of the quartz plate.

<Evaluation method>
Evaluation of the optical isomer resolution of the chiral stationary phase using the evaluation cell 7 can be performed as follows.
The solution tank 6 is filled with a solution containing the optical isomer to be measured, and the crystal unit 1 of the sensor unit 5 is immersed in the solution, and the crystal plate 2 is transferred from the first electrode 3a and the second electrode 3b. When a voltage is applied, the crystal plate 2 is deformed, and the crystal resonator 1 vibrates at a frequency determined by the shape and size of the crystal plate 2. At this time, when the optical isomer is bound to the active substance by interaction with the active substance on the first electrode 3a, the crystal is changed according to the amount of the optical isomer bound over time, that is, the mass of the optical isomer. The frequency of vibration of the vibrator 1 is lowered. Since the amount of optical isomers to bind depends on the strength of interaction between the active substance and the optical isomer, other optical isomers of the chiral molecule that are not detected are detected by the optical isomer to be detected. If an active substance that interacts more strongly is used, the frequency decrease width when the optical isomer to be detected binds is larger than the frequency decrease width when another optical isomer binds.

Therefore, when a difference in the decrease in frequency occurs, it can be confirmed that the active substance can be separated by optical HPLC by HPLC when used in the chiral stationary phase of the chiral column. In addition, the elution time in HPLC is delayed when the frequency decrease is larger than the frequency decrease, and the difference in elution time in HPLC increases as the difference in frequency decrease increases.
As described above, by measuring the frequency fluctuation of the piezoelectric element due to the interaction between the optical isomer and the active substance, it is possible to evaluate the resolution of the optical isomer as the chiral stationary phase of the active substance.

  Here, the case where the crystal unit 1 is vibrated after the sensor unit 5 is immersed in the solution containing the optical isomer has been described. For example, the sensor unit may be included in the solution that does not contain the optical isomer. After immersing 5, the crystal unit 1 may be vibrated, and then an optical isomer may be added to the solution.

  It is preferable that the optical isomer to be used for the measurement of frequency fluctuation once is not a mixture of a plurality of types of optical isomers but one type. That is, for multiple types of optical isomers to be measured, each type is subjected to sequential measurement to obtain frequency fluctuation data individually, and the optical isomers to be detected and other optical isomers to be separated on a chiral column. It is preferable to compare data between.

  The detection sensitivity of the optical isomer is improved as the vibration frequency of the crystal resonator 1 is increased, but an appropriate frequency may be appropriately set in consideration of the type of the active substance, and the preferable range thereof is first. As stated. Since the frequency of the crystal unit 1 is determined by the shape and size of the crystal unit 2, if the frequency of the crystal unit 1 is to be changed, the crystal unit 2 having a different shape and / or size may be used.

  The composition of the solution to be subjected to the measurement and the temperature of the solution at the time of measurement are preferably selected in consideration of the composition of the mobile phase used at the time of separation by the chiral column and the set temperature. The closer the solution composition excluding the optical isomers at the time of measurement is to the above mobile phase composition, and the closer the temperature of the solution at the time of measurement is to the set temperature of the mobile phase, the more faithfully the resolution evaluation results will be closer to the separation results by the chiral column Reflected.

In other words, the composition of the solution used for the measurement is preferably one that can be used as a mobile phase during separation by a chiral column.
Moreover, 15-40 degreeC is preferable and the temperature of the solution at the time of a measurement has more preferable 25-37 degreeC.

  The sensor unit 5 is preferably stabilized before measurement of frequency fluctuations. For example, the sensor unit 5 is preferably immersed in a solvent of the same type as the solvent component of the solution to be used for measurement for a predetermined time. The temperature of the solvent at this time may be the same as the temperature of the solution at the time of measurement. The immersion time is preferably 5 to 40 minutes, more preferably 10 to 30 minutes, and particularly preferably 15 to 20 minutes.

When comparing data on frequency fluctuations, it is preferable that the optical isomers used for measurement have the same concentration in the solution. By doing so, the accuracy of data comparison can be improved.
And it is preferable that the density | concentration in the solution of the optical isomer used for a measurement is 0.1-10 mg / mL.
Further, the solvent of the solution containing the optical isomer may be any solvent that dissolves the optical isomer, and may be appropriately selected according to the type of the optical isomer. Among them, preferable examples include alcohols such as methanol and ethanol, and buffers that can be used as a mobile phase at the time of separation using a chiral column, and ethanol is particularly preferable.

  Furthermore, the concentration of the optical isomer to be used for measurement in the solution tank 6 is preferably applicable as the concentration in the mobile phase at the time of separation by the chiral column, and more preferably 1 to 1000 μg / mL.

When the evaluation cell 7 includes a plurality of sensor parts 5 and the active substances fixed to the first electrodes 3a of these sensor parts 5 are all different, the interaction between these active substances and optical isomers A decrease in frequency is observed in all the sensor units 5 with a decrease width corresponding to the strength.
That is, by using a plurality of types of active substances simultaneously for measurement, an optimal active substance for a chiral stationary phase can be selected in a shorter time.
On the other hand, when the active substances fixed to the first electrodes 3a of the plurality of sensor units 5 are all the same, the degree of variation in the observed frequency decrease width was examined, and used for the chiral stationary phase. In this case, the reproducibility of the optical isomer separation of the active substance can be confirmed.

  When the material of the solution tank 6 is transparent, as described above, more detailed measurement can be performed by combining optical analysis such as measurement of solution absorbance, chemiluminescence, or fluorescence. In particular, when the evaluation cell 7 includes a plurality of sensor units 5, more information can be obtained by observing simultaneously the frequency variation of the first electrode 3a of each sensor unit and the optical properties of the solution in the vicinity of the electrode. It can be obtained and a highly accurate evaluation result can be obtained.

After the evaluation is completed, the first electrode 3a can be regenerated by washing the first electrode 3a and removing the fixed active substance. The sensor unit 5 on which the first electrode 3a is regenerated can be used for evaluating different chiral stationary phases. Here, “regeneration” means that no substance is bonded on the first electrode 3a.
The cleaning of the first electrode 3a may be performed by the above-described method of removing foreign matters from the fixed surface of the electrode and flattening the fixed surface.

In addition, as described above, when the active substance is immobilized to the electrode via a non-covalent bond, for example, as in the method involving biotin-avidin complex formation, the non-covalent bond can be eliminated. The active substance can be removed, and it can be used again for the evaluation of the chiral stationary phase without regenerating the first electrode 3a. In this case, the active substance can be re-immobilized via a non-covalent bond, but this technique is particularly suitable for continuous measurements using different active substances.
The noncovalent bond can be eliminated by adjusting the pH of the solution in which the sensor unit 5 is immersed, for example.

<Evaluation equipment>
FIG. 2 is a schematic configuration diagram illustrating an evaluation apparatus including the evaluation cell, and FIG. 3 is a perspective view illustrating the evaluation cell.
The evaluation apparatus 20 shown here can repeatedly measure frequency fluctuations caused by the interaction between the optical isomer and the active substance while washing the evaluation cell.
Reference numeral 21 denotes the evaluation cell described above. And as shown to Fig.3 (a), the solution tank 211 is a rectangular parallelepiped flow cell type in which the inflow port 211a for making a solution flow in, and the discharge port 211b for making it discharge | emit are provided separately. In the solution tank 211, a sensor unit 8 is immersed in which three sensor parts 5a, 5b and 5c, each having a different active substance, are arranged on the same surface of the resin plate. That is, the sensor unit 8 shown here can measure the frequency fluctuations of the crystal resonators at the same time for the three sensor units 5a, 5b and 5c with a single supply of the solution containing the optical isomer to be detected. is there.

In the evaluation cell 21 shown in FIG. 3A, in the three sensor parts 5a, 5b and 5c of the sensor unit 8, the electrode surfaces on which the active substance is fixed are exposed so as to face in the same direction. Yes.
The sensor unit 8 has a surface opposite to the surface on which the sensor portions 5a, 5b and 5c are disposed (hereinafter abbreviated as a non-sensor surface) in contact with the bottom surface 211c of the solution tank 211 so that it can be attached and detached. It is installed as possible.
And in the sensor unit 8, the three sensor parts 5a, 5b, and 5c are arrange | positioned in series in the substantially the same direction as the moving direction of the solution shown by the arrow in the figure.

The sensor unit 8 does not necessarily need to be detachable from the solution tank 211, for example, is fixed using an adhesive. However, the sensor unit 8 is detachable because it is easy to maintain and clean. It is preferable. The method of detachably installing may be a known method. For example, a protrusion is provided on one of the solution tank 211 and the sensor unit 8, a recess is provided on the other, and the protrusion and the recess are engaged with each other in a detachable manner. .
Further, the resin plate of the sensor unit 8 may be made of other insulating material such as a glass plate.

The installation form of the sensor unit 8 is not limited to the one shown here, and can be selected as appropriate. FIGS. 3B and 3C are perspective views illustrating other installation forms.
In (b), the sensor unit 8 is installed upright on the bottom surface 211c of the solution tank 211 without contacting the side surface. In the three sensor parts 5a, 5b and 5c, the electrode surfaces on which the active substance is fixed are exposed so that they all face the same direction and have the same height from the bottom surface 211c of the solution tank 211. . And these sensor parts are arrange | positioned in series in the substantially the same direction as the moving direction of the solution shown by the arrow.
Although not shown here, for example, the direction of the electrode surface on which the active substance is fixed is the same as in the case of FIG. 3B, and the non-sensor surface is placed in contact with the side surface 211e of the solution tank 211. May be. In this case, the sensor unit 8 may or may not stand upright on the bottom surface 211c of the solution tank 211. Further, instead of the bottom surface 211c, it may be provided upright from the top surface 211d.

  In (c), the sensor unit 8 is similar to (a) except that the three sensor units 5a, 5b and 5c are arranged in series in a direction substantially perpendicular to the moving direction of the solution. It is installed on the bottom surface 211c.

  Although FIG. 3 shows an example in which the number of sensor units 8 installed in the solution tank 211 is one, a plurality of sensors may be installed. In this case, the arrangement form of the plurality of sensor units 8 can be selected as appropriate. As a preferable arrangement form, for example, the electrode surfaces on which the active substance is fixed are arranged so as to face each other, or in directions orthogonal to each other. For example, the one installed in the same direction can be exemplified. Alternatively, the plurality of sensor units 8 may be arranged so that the electrode surfaces on which the active substance is fixed are all located in the same plane, or are arranged in planes parallel to each other. It doesn't have to be either.

  Here, an example is shown in which the number of sensor units installed in the sensor unit 8 is three, but the number of installations is not limited to this, and can be appropriately selected according to the purpose. Evaluation can be performed more efficiently by increasing the number of installations within a range that does not impair the effects of the present invention.

  In addition, here, an example using the sensor unit 8 in which a plurality of types of sensor units are installed is shown, but the evaluation cell is not limited to this, for example, a plurality of types of sensors without using the sensor unit. May be arranged in the solution tank 211. In this case, the installation methods and arrangement forms of the plurality of types of sensor units may be the same as the installation methods and arrangement forms of the plurality of sensor units 8 described above.

  As shown in FIG. 2, in the evaluation cell 21, the sensor units 5a, 5b and 5c are electrically connected to an oscillation circuit 22 for applying a voltage between the electrodes to vibrate the crystal resonator (FIG. 3). Further, the frequency measurement device 23 for measuring the vibration frequency of the crystal resonator is electrically connected to the oscillation circuit 22. The frequency measurement device 23 is connected to a computer 24 and a display 24 that automatically perform information processing related to frequency fluctuation measurement data and display the result as external equipment. The information processing referred to here includes recording and comparison of measurement data.

  Further, the measurement buffer 291 supplied to the evaluation cell 21 and the cleaning liquid 292 for cleaning the evaluation cell 21 are separately filled in containers, and these containers are connected to the switching valve 26 through piping. The switching valve 26 is connected to a pump 25 through a pipe, and the pump 25 is connected to the solution tank 211 through a pipe and an inlet 211a. That is, by operating the switching valve 26, either the measurement buffer solution 291 or the cleaning solution 292 can be supplied to the evaluation cell 21. As the measurement buffer solution 291, a buffer solution that can be used as a mobile phase at the time of separation using a chiral column can be exemplified.

  On the other hand, a container filled with a solution 293 containing an optical isomer to be measured (hereinafter abbreviated as measurement liquid) is connected to the open / close valve 27 through a pipe, and the open / close valve 27 is connected to the open / close valve 27 through the pipe from the pump 25. On the upstream side, it is connected to a pipe for supplying the measurement buffer 291 or the cleaning liquid 292. That is, when the opening / closing valve 27 is opened, the measurement liquid 293 can be supplied to the evaluation cell 21 using the measurement buffer 291 as a mobile phase.

  The discharge port 211b of the solution tank 211 is connected to the switching valve 26 through a pipe. By switching the switching valve 26, the liquid whose measurement has been completed is extracted in two paths, and the first waste liquid 294 and the second waste liquid 294 are respectively extracted. The waste liquid 295 can be separately collected in a container. Therefore, for example, the used measurement buffer solution 291 or measurement solution 293 and the used cleaning solution 292 are separately collected, and the used measurement buffer solution 291 or measurement solution 293 is discarded as it is, and the cleaning solution is used. 292 may be reused. In this case, one path may be connected to a container filled with the cleaning liquid 292 so that the cleaning liquid 292 can be recycled.

  In addition, although illustration is abbreviate | omitted here, you may make it the operation of the pump 25, the switching valve 26, and the on-off valve 27 be controlled automatically by the computer connected to these.

Further, as described above, when the active substance is fixed to the electrode through a non-covalent bond, a buffer solution or the like that can eliminate this non-covalent bond is used as the washing liquid 292, so that the electrode can be used after the measurement is completed. The active substance can be removed from. Further, for example, instead of the measurement solution 293, a solution containing an agent bound with avidin, biotin, or the like is used, and the solution is supplied to the evaluation cell 21 so that the agent can be removed via a non-covalent bond. Can be fixed to the electrode.
In this way, the removal and fixation of the active substance can be performed automatically. For example, the sensor part that fixes the active substance to the electrode via a covalent bond and the active substance is fixed to the electrode via a non-covalent bond. Non-covalent bond when using an evaluation cell that is used in combination with a sensor unit, or when using an evaluation cell that uses multiple types of sensor units that can be formed and eliminated under conditions where non-covalent bonds are different from each other. It is also possible to continuously switch the combination of the sensor parts in the evaluation cell 21 by sequentially changing the type of the active substance to be fixed via the. In this case, the evaluation can be performed more efficiently.

  When evaluating the optical isomer resolution using the evaluation device 20, the measurement solution 293 is supplied to the evaluation cell 21 together with the measurement buffer 291 that is a mobile phase, and the frequency of the crystal resonator is supplied by the frequency measurement device 23. What is necessary is just to measure a fluctuation | variation for every sensor part. Such measurement is performed by sequentially using a solution containing the optical isomer to be detected and a solution containing other optical isomers as the measurement solution 293, and the difference in the frequency reduction range is the largest among the optical isomers. If the sensor part is specified, the active substance of the sensor part interacts most strongly with the optical isomer to be detected. Therefore, when used for a chiral stationary phase, the optical isomer separation ability is the highest. Can be determined to be high. After the measurement is completed, the evaluation cell 21 can be cleaned with the cleaning liquid 292.

  At this time, for example, if the operation of the pump 25, the switching valve 26 and the opening / closing valve 27 is automatically controlled to automatically supply the measurement buffer solution 291, the measurement solution 293, and the cleaning solution 292, the measurement and All information processing related to measurement data can be performed automatically. Therefore, when performing the evaluation, the operator only needs to prepare the measurement buffer solution 291, the cleaning solution 292 and the measurement solution 293, fill the container, and initially set the measurement conditions. The evaluation apparatus 20 has a simple measurement operation and can be evaluated quickly, and the supply and collection paths of the measurement buffer solution 291, the washing solution 292, and the measurement solution 293 are airtight and separated from the outside as necessary. can do. Therefore, for example, when the measurement liquid 293 contains a component that is extremely harmful to the human body, the exposure time of the operator to the measurement liquid 293 can be significantly reduced, which is useful for ensuring the safety of the worker. is there.

Hereinafter, the present invention will be described in more detail with reference to specific examples. However, the present invention is not limited to the following examples.
[Example 1]
((S) -naproxen and (R) -naproxen are used as optical isomers, and a compound represented by the following chemical formula (1) (cyclomaltoheptosyl- (6 → 1) -O-α-D-glucopyranosyl as an active substance) -Evaluation using (4-> 1) -O-α-D-glucopyranoside uronic acid)
Using the evaluation cell shown in FIG. 1, the active substance was fixed to the electrode according to the following procedure, and a sensor part was produced. As the crystal resonator, a crystal plate having a diameter of 8.7 mm and a thickness of 60 μm and a gold electrode having a diameter of 2.5 mm and a thickness of 260 nm was used.
40 μL of 5.0 mM aminoethanethiol aqueous solution was dropped on the gold electrode part of the sensor part, and allowed to stand for 30 minutes at room temperature / humidity to perform SAM formation. Utilizing this, 400 μL of a dimethyl sulfoxide solution of an active substance represented by the following formula (1) of 3.0 mg / mL, 40 μL of 100 mg / mL N-hydroxysuccinimide and 100 mg / mL 1-ethyl-3- (Dimethylaminopropyl) carbodiimide (40 μL) was added, and the mixture was allowed to stand at room temperature / humidity for 30 minutes. 40 μL of the active substance thus activated was dropped onto the gold electrode part of the sensor part, and allowed to stand for 1 hour at room temperature / humidity.
And the sensor part was immersed in 8.0 mL purified water with which the container-type solution tank with a capacity | capacitance of 11 mL was filled, and was stabilized at 25 degreeC for 15 to 20 minutes. Next, the gold electrode was vibrated at 27 MHz, and 8.0 μL of an ethanol solution containing (S) -naproxen to be measured at a concentration of 5 mg / mL was injected, and the change in frequency was measured. Similarly, (R) -naproxen was also measured. The results are shown in FIG.
As is clear from FIG. 4, the average value of the frequency change 4 minutes after the start of the measurement is (S) -naproxen is −50.5 Hz, (R) -naproxen is −70.1 Hz, and (S Significant differences were observed between) -naproxen and (R) -naproxen.
In fact, the strength of the interaction between naproxen and the active substance is larger in the (R) -form than in the (S) -form, and a commercially available HPLC chiral column using the same active substance as the chiral stationary phase is used. When the separation was performed, the (R) -form was more consistent with the elution later than the (S) -form.

[Example 2]
(D-acetylphenylalanine and L-acetylphenylalanine as optical isomers, and compound (N-[(R) -1- (α-naphthyl) ethylaminocarbonyl] -L represented by the following chemical formula (2) as an active substance) -Evaluation using tert-leucine)
Evaluation was performed in the same manner as in Example 1 except that the above compound was used as an optical isomer and an active substance. The results are shown in FIG.
As is clear from FIG. 5, the average value of the frequency change 4 minutes after the start of measurement is -59.5 Hz for D-acetylphenylalanine, -73.4 Hz for L-acetylphenylalanine, and D-acetylphenylalanine. A significant difference was confirmed between L and acetylphenylalanine.
In fact, the strength of the interaction between phenylalanine and the active substance is greater in the L-form than in the D-form, and separation can be performed using a commercially available chiral column for HPLC using the same active substance as the chiral stationary phase. When performed, the L-form was in good agreement with the elution later than the D-form.

[Example 3]
((R) -1,1′-bi-2-naphthol and (S) -1,1′-bi-2-naphthol as optical isomers, and a compound represented by the following chemical formula (3) as an active substance ( Evaluation using N- (3,5-dinitrophenylaminocarbonyl) -D-phenylglycine)
Evaluation was performed in the same manner as in Example 1 except that the above compound was used as an optical isomer and an active substance. The results are shown in FIG.
As is clear from FIG. 6, the average value of the frequency change 2 minutes after the start of measurement is (R) -1,1′-bi-2-naphthol is −27.9 Hz, and (S) −1,1 '-Bi-2-naphthol is -59.5 Hz, significant between (R) -1,1'-bi-2-naphthol and (S) -1,1'-bi-2-naphthol The difference was confirmed.
Actually, the strength of the interaction between 1,1′-bi-2-naphthol and the active substance is larger in the (S) -form than in the (R) -form, and the same active substance is treated with a chiral stationary phase. When the separation was carried out using the commercially available HPLC chiral column used in (1), the (S) -isomer was more consistent with the elution later than the (R) -isomer.

  The present invention can be used for chiral separation of optical isomers, and is particularly useful for development of pharmaceuticals.

It is a figure which illustrates the cell for evaluation used by the present invention, (a) is a perspective view in the state where the sensor part was immersed in the solution in a solution tank, (b) is a front view, (c) is A It is sectional drawing in the -A line. It is a schematic block diagram which illustrates the evaluation apparatus of this invention. It is a perspective view which illustrates other embodiment of the cell for evaluation used by the present invention. 4 is a graph showing a change in frequency of a crystal resonator in Example 1. 6 is a graph showing a frequency change of a crystal resonator in Example 2. 10 is a graph showing a change in frequency of a crystal resonator in Example 3.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Quartz crystal resonator, 2 ... Quartz plate, 3 ... Electrode, 3a ... 1st electrode, 3b ... 2nd electrode, 5 ... Sensor part, 6 ... Solution tank 7 ... Evaluation cells

Claims (8)

A method for evaluating the optical isomer resolution of a chiral stationary phase for separating and detecting optical isomers to be detected,
An agent for the chiral stationary phase, which interacts with the optical isomer to be detected, is immobilized on one or both electrodes of the piezoelectric element,
A linking substance that is fixed to the electrode at one end and a main chain having a carboxyl group at the other end is bound to avidin, the agent having an amino group is bound to biotin, and biotin- By forming an avidin complex, the active substance is fixed to the electrode through a non-covalent bond, and the active substance is fixed to the electrode and removed from the electrode by forming and eliminating the non-covalent bond. Is possible,
An evaluation cell comprising: a sensor unit capable of being electrically connected to a circuit for applying a voltage between the electrodes and a frequency measuring device; and a solution tank for immersing the sensor unit in a liquid. Use
In the solution containing the optical isomer to be detected in the solution tank, the interaction between the optical isomer to be detected and the active substance while applying a voltage between the electrodes of the sensor unit to vibrate the piezoelectric element. The frequency variation of the piezoelectric element caused by the above is measured, and the frequency variation is measured using a solution containing an optical isomer that is not detected instead of the solution, and the obtained measurement results are compared. A method for evaluating the resolution of optical isomers. A method for evaluating the optical isomer resolution of a chiral stationary phase for separating and detecting optical isomers to be detected,
An agent for the chiral stationary phase, which interacts with the optical isomer to be detected, is immobilized on one or both electrodes of the piezoelectric element,
A linking substance in which the main chain having an amino group at one end is fixed to the electrode and the main chain having a linear chain is bonded to biotin, the active substance having a carboxyl group is bonded to avidin, and biotin- By forming an avidin complex, the active substance is fixed to the electrode through a non-covalent bond, and the active substance is fixed to the electrode and removed from the electrode by forming and eliminating the non-covalent bond. Is possible,
An evaluation cell comprising: a sensor unit capable of being electrically connected to a circuit for applying a voltage between the electrodes and a frequency measuring device; and a solution tank for immersing the sensor unit in a liquid. Use
In the solution containing the optical isomer to be detected in the solution tank, the interaction between the optical isomer to be detected and the active substance while applying a voltage between the electrodes of the sensor unit to vibrate the piezoelectric element. The frequency variation of the piezoelectric element caused by the above is measured, and the frequency variation is measured using a solution containing an optical isomer that is not detected instead of the solution, and the obtained measurement results are compared. A method for evaluating the resolution of optical isomers. The electrode is a gold electrode, the connecting material is a linear carboxyalkanethiol, carboxyalkenethiol, or carboxyalkynethiol, and the connecting material has a sulfur atom bonded to the gold electrode surface. The method for evaluating optical isomer resolution according to claim 1. The electrode is a gold electrode, the connecting material is a linear aminoalkanethiol, aminoalkenethiol, or aminoalkynethiol, and the connecting material has a sulfur atom bonded to the gold electrode surface. The method for evaluating optical isomer resolution according to claim 2. The method for evaluating optical isomer resolution according to any one of claims 1 to 4, wherein the piezoelectric element is a crystal resonator. Evaluation method for optical isomer separation capability according to claim 4 or 5, wherein the number of carbon atoms of the amino alkanethiol is 1-4. An evaluation apparatus for use in the method for evaluating optical isomer resolution according to any one of claims 1 and 3-6 ,
One or both electrodes of the piezoelectric element are provided with a sensor part in which an agent for chiral stationary phase that interacts with an optical isomer to be detected is fixed, and a solution tank in which the sensor part is immersed in a liquid. An evaluation cell is provided, and the evaluation cell includes a plurality of types of the sensor units having different active substances, and the electrodes are electrically connected to a circuit and a frequency measuring device for applying a voltage between the electrodes. And a solution supply means for supplying the solution to the evaluation cell ,
A linking substance that is fixed to the electrode at one end and a main chain having a carboxyl group at the other end is bound to avidin, the agent having an amino group is bound to biotin, and biotin- By forming an avidin complex, the active substance is fixed to the electrode through a non-covalent bond, and the active substance is fixed to the electrode and removed from the electrode by forming and eliminating the non-covalent bond. evaluation device optical isomers resolution characterized that you have been and can be. An evaluation apparatus used in the evaluation method of the optical isomers resolution according to any one of claims 2-6,
One or both electrodes of the piezoelectric element are provided with a sensor part in which an agent for chiral stationary phase that interacts with an optical isomer to be detected is fixed, and a solution tank in which the sensor part is immersed in a liquid. An evaluation cell is provided, and the evaluation cell includes a plurality of types of the sensor units having different active substances, and the electrodes are electrically connected to a circuit and a frequency measuring device for applying a voltage between the electrodes. And a solution supply means for supplying the solution to the evaluation cell ,
A linking substance in which the main chain having an amino group at one end is fixed to the electrode and the main chain having a linear chain is bonded to biotin, the active substance having a carboxyl group is bonded to avidin, and biotin- By forming an avidin complex, the active substance is fixed to the electrode through a non-covalent bond, and the active substance is fixed to the electrode and removed from the electrode by forming and eliminating the non-covalent bond. evaluation device optical isomers resolution characterized that you have been and can be.

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