JPH02269953A - Capillary electrophoresis device - Google Patents

Abstract

PURPOSE:To obtain the automated device which is good in reproducibility by housing a capillary tube, sample microinjector, waste liquid selector valve, and detector for detecting separated sample components in a thermostatic chamber. CONSTITUTION:A buffer soln. tank 3 for supplying and circulating the buffer soln. at a specified level, the sample microinjector 10 which is connected to a solvent selector valve 19 for selecting and supplying an initial packing liquid and a cleaning liquid and can change the inside volume and the waste liquid selector valve 4 which discharges the liquid remaining in the capillary tube 1 are provided. The capillary tube 1, the injector 10, the valve 4 and the detector 6 for detecting the separated sample components are housed in the thermostatic chamber 16. The valve 19 is selected to supply the initial packing liquid or the cleaning liquid into the injector 10 and the valve 4 is selected when the liquid is filled in the capillary tube 1 by the injector 10. Sequential control is so executed that the valve 4 and the valves 10a, 10b in the injector 10 are switched when the specified amt. of the sample is injected into the tube by the injector 10 and thereafter, a switch 11 is closed at the specified timing.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention is directed to capillary electrophoresis (capillary electrophoresis).
ryeelectrophoresis, CB) equipment, electrokinetic chromatography (electrok)
inetic chronatography, EKC
) device, capillary chromatography using electroosmotic flow (capillary chromatography)
d chrom+natography witiel
The present invention relates to a capillary electrophoresis device with excellent reproducibility that can be commonly used as a capillary electrophoresis device and a capillary isotachophoresis device.

<Conventional technology> Capillary electrophoresis, electrokinetic chromatography,
Capillary liquid chromatography, which uses electroosmotic flow, and isotacho are both types of electrophoretic analysis methods that perform separation analysis using differences in the electrical mobility of a sample under high electric field. This method differs greatly from other electrophoresis methods in that separation is performed in a hollow tube (capillary tube) with an inner diameter of several hundred μm or less. These methods basically consist of a hollow capillary column and a high-voltage power supply, and each of these methods can be used in the same device.

In other words, capillary electrophoresis is a method in which a high voltage is applied to a capillary tube filled with MI buffer, and separation is performed using the difference in charge (difference in ionic strength) between the sample components to be measured. This is an improved version of the zonal pneumophoresis method that has been used since then.

Electrokinetic chromatography, in a broad sense, is a separation method that utilizes electrokinetic phenomena in a capillary tube to which a high voltage is applied, but in a narrower sense, it is a method of separation using micelle solubilization proposed by Maebe et al. Refers to electrokinetic chroma 1-graphy using M E CC (ll
1celler electrokinetic ca
It is also called pillary chloratography.

The major difference between capillary electrophoresis and electrokinetic chromatography is that electrokinetic chromatography performs separation using electroosmotic flow caused by ionic groups on the surface of the capillary tube. . That is, a buffer solution containing an ionic surfactant is used as the liquid filling the capillary. In addition, the sample injected into the capillary is taken up by surfactant micelles contained in the solvent and moves through the capillary on the electroosmotic flow. The micelles move in the direction opposite to the charge on the micelles, but if the electroosmotic flow and the electrophoresis of the micelles are in opposite directions, the electrophoretic mobility between the micelles containing each component of the sample will change. The difference can be magnified by the electroosmotic flow flowing in the opposite direction, resulting in a lick-heavy section. Therefore, using capillary electrophoresis, it is also possible to separate nonionic hydrophobic substances.

Gearpilary liquid chroma 1-graphie using electroosmotic flow is a method of separation that actively utilizes electroosmotic flow, similar to the above-mentioned electrokinetic chroma 1-graphie, but it is different from pure electrophoresis. .

In such capillary/j support chromatography using electroosmotic flow, the main role of electroosmotic flow is as a liquid pump for the mobile phase in liquid chromatography. The sample moves through the capillary using electroosmotic flow, and is separated by interaction with the functional groups on the inner surface of the capillary. However, depending on the properties of the sample and the combination of mobile phases, separation may be performed using capillary electrophoresis or electrokinetic chromatography.

On the other hand, isotacho is one of the pure electrophoresis methods, but it differs greatly from the above three methods.

The reason is that in the above three methods, one type of solvent (buffer solution) is used, but in Isotaco, two types of buffer solutions are used.

That is, the first H buffer solution is called a leading liquid and is made up of ions having higher mobility than the ions to be measured, and is filled into the capillary in advance before sample injection. The second IJj liquid is called a tailing liquid and is made up of ions with properties completely opposite to those of the leading liquid, that is, ions having a lower mobility than all the ions to be measured, and is placed immediately behind the sample after the sample is injected. The sample is analyzed while being sandwiched between the above two M solution, and the separation is also carried out between these two solutions.
It is carried out between two ftl solutions.

By the way, although devices for capillary electrophoresis have been sold on the market in recent years, there is no device that can be used in combination with isotacho.
Automation has not progressed, and many problems remain in terms of measurement reproducibility. In the above-mentioned method, there are problems such as the following (1) to (2) in order to perform analysis with good reproducibility. DO: ■ A constant, extremely small amount of sample is always injected into the capillary tube.

■The time from sample injection to the start of analysis must be controlled at a constant level.

■Reducing the effects of Joule heat caused by the application of high voltage.

■ Avoid being affected by gases generated by electrolysis.

■The inner surface of the capillary tube and the electrode surface should not be contaminated by samples, etc.

On the other hand, conventional injection methods include injection using the difference in the water level of the sample solution (head difference), injection using electrophoresis and electroosmotic flow, and injection using split 1 (in actual practice). 161355/1983> etc.The method of injection using the difference in the water level of the sample solution (head difference) is as follows.
Because it is a simple method and it is difficult to control head differences, good reproducibility cannot be obtained. In the injection method using electrophoresis and electroosmotic flow, the composition of the injected sample differs from the original sample because the mobility differs depending on the sample. Although the split injection method is easy to automate, good reproducibility may not be obtained due to sample viscosity, contamination of the sugerite part, etc. In order to address the above-mentioned problems regarding automation and reproducibility, an injection method using a variable volume microvalve has been proposed.

(Unexamined Japanese Patent Publication No. 63-253247) This method is the best injection method at present because it is easy to automate and shows good reproducibility, but there are problems with RWI when using it for isotacho.

In electrophoresis, the reproducibility of analysis is greatly impaired by the generation of Joule heat, but in electrophoresis using capillary tubes, the effect is very noticeable. However, at present, sufficient measures have not been taken to address this problem.

In addition, in capillary electrophoresis, if gas generated by electrolysis enters the capillary tube, the current is interrupted and measurement is interrupted. As a countermeasure to this problem, the buffer solution is refluxed and the inlet of the capillary tube is constantly closed. This problem can be solved by washing out the generated gas.

Furthermore, contamination of the inner surface of the capillary tube by injected sample components and contamination of the electrode surface by sample components (absorption)
4) also seriously impairs the reproducibility of analysis, and at present there are almost no countermeasures taken against these.

゛〈Problems to be solved by the invention〉 The technical problems to be solved by the present invention are the above-mentioned 4.
Automated capillary electrophoresis with good reproducibility that can be commonly used as a capillary electrophoresis device, an electrokinetic chromatography device, a capillary liquid chromatography device using electroosmotic flow, and an isotacho device. The goal is to provide equipment.

Means for Solving the Problems> The features of the present invention that solve the above-mentioned problems include means for supplying and circulating the #1 buffer solution at a constant water level in a capillary electrophoresis device, and a means for supplying and circulating the #1 buffer solution at a constant water level, and a method for supplying and circulating the initial filling solution and washing solution. A micro-sample injection device that has a fluid switching valve that can switch to supply one or the other to change the internal volume, and a waste fluid switching valve that drains the fluid remaining in the capillary tube before or after the start of analysis. and the capillary tube and the microsample injection device.

The solvent switching valve and a detector for detecting the separated sample components are housed in a constant temperature bath, and switch means for applying the high voltage power source is provided to both ends of the capillary tube, and the solvent switching valve is installed. When the capillary tube is filled with the initial filling liquid or washing liquid by the micro sample injection device, the waste liquid switching valve is switched to supply the initial filling liquid or washing liquid to the micro sample injection device, and the waste liquid switching valve is switched to When a certain amount of sample is injected by the sample injector, the waste liquid switching valve and the valve of the micro sample injector are switched, and then sequence control is performed so that the switch is closed at a certain timing, thereby solving the problem. This is the solution.

Furthermore, the present invention connects the positive and negative sides of a high-voltage DC power source to both ends of a capillary tube filled with ui solution, and uses electrokinetic phenomena to control the sample injected into the capillary tube. and separating the sample components,
In the analyzer for qualitative and quantitative analysis of the sample components,
A micro-sample injection device that has a means for supplying and circulating a buffer solution at a constant water level, a means for supplying an initial filling liquid, and can change the internal volume, the capillary tube, the micro-sample injection device, and the separated sample components. A detector for detecting is housed in a constant temperature bath, and switch means for applying the high voltage to both ends of the capillary tube is provided, and the initial filling liquid is supplied to the micro sample injection device, and the The above problem is similarly solved by sequence control so that the switch closes at a certain timing when a certain amount of sample is injected into the capillary tube by a microsample injection device.

Function> The technical means related to the present invention functions as follows. In other words, by adopting a flow path that supplies and circulates the buffer solution at a constant water level (head pressure) and a micro sample injection device that does not have a flow path that supplies initial filling liquid, etc., and whose volume can be changed, it is possible to It can be used for both electrophoresis and isotacho measurements. Furthermore, since the sample injection device injects the sample using a buffer solution at a constant pressure, the composition of the sample components hardly changes.
To prevent stains on the electrodes, since the buffer pipe itself on the sample injection device side is used as an electrode, it can be constantly cleaned with the circulating buffer solution. This circulation of the buffer solution also serves to push out the bubbles generated by electrolysis near the electrodes to the outside of the device.

``Improvements in measurement reproducibility due to the generation of Joule heat'' include the capillary tube, the microsample injection device,
By housing the waste liquid switching valve and the detector for detecting the separated sample components in a thermostatic chamber, the Joule heat generated by the application of the high voltage can be dissipated well. The tube portion is always kept at a constant temperature, and the temperature of the injected sample is also constant, resulting in good measurement reproducibility. Also·
Since the detector is also installed in a constant temperature chamber, it is not affected by the external environment and enables highly sensitive measurements.

Furthermore, injection of initial filling liquid, injection of sample, application of high voltage,
All capillary tube cleaning and other operations are controlled by a sequencer, allowing for highly reproducible injection and measurement.

<Example> Hereinafter, the present invention will be described in detail using figures. FIG. 1 is an explanatory diagram of the configuration of an embodiment of the present invention. In the figure, 8 is a constant water level creator, two pipes 8a and 8b are connected in the middle, and a pump 8C is operated from the bottom of the pipe 8a. The buffer solution stored in the buffer solution tank 8d is fed into the buffer solution tank 8d. In addition, the solvent that overflowed in the tube 8a
b, and is then refluxed back to the buffer tank 8d. 17 is an initial filling liquid tank that stores initial filling liquid, and 18 is a cleaning liquid tank that stores cleaning liquid 21. Further, these tanks 17, 18 can be switched by one solvent switching valve. Therefore, the initial light #4 liquid tank 1 is passed through the sample injection valve 10.
7#JL< is the initial filling liquid (solvent B) due to the head difference that occurs between the flow path from the cleaning liquid tank 18 and the capillary tube 1, as in the case of the constant water level creator 8. Alternatively, the cleaning liquid is sent to the capillary tube 1. Further, 9 is a microsyringe for supplying a sample, and 10 is a sample injection valve for injecting a minute amount of sample into the capillary tube 1. Reference numeral 4 denotes a waste liquid switching valve, which connects the capillary tube 1 to the W soaking liquid tank 3 during measurement of sample components, but is switched to the waste liquid side when the initial filling liquid or cleaning liquid flows through the capillary tube 1. 1
1 is a switch that applies a high voltage E to the capillary tube 1.

12 sends a control signal to the sample injection valve 4 according to a predetermined sequence. This is a sequencer that supplies the switch 11iK-4 processing unit 13. Note that 14 is an ammeter, 15 is a recorder, and 16 is a constant temperature bath.

The operation of the present invention will be explained below. In FIG. 1, first, the target first slow Wt liquid A is poured into the constant water level generator 8.
Sera 1-shi is placed in the buffer tank 8d of the buffer solution tank 8d, the circulation pump 10c is operated, and Ml fM ? Fill the tank 10a with uiMA. Next, fill the target second buffer B with the initial filling liquid tank 17 and the lower M8ir? & Set it in tank 3. If the inside of the capillary tube 1 needs to be cleaned, a cleaning liquid is set in the cleaning liquid tank 18.

After that, the sample injection valve 10 is switched and connected to the initial filling liquid side, the solvent switching valve 19 is connected to the initial filling liquid side, the waste liquid switching valve 4 is switched to the waste liquid side, and the inside of the capillary tube 1 is filled with the initial filling liquid. figure. At this time, the sample is injected into the measuring hole in the sample injection valve 10 using a microsyringe or an autosampler.

The sample injection valve 10 is switched to the sample injection side, and the waste liquid switching valve 4 is switched to the buffer tank 3 side. A fixed amount of the sample is injected into the capillary tube 1 by the hydraulic pressure from the constant water level generator 8.

The injection amount is determined when the above measurement hole is inserted into the capillary tube 1 [
The time determined by the connection to the 1 side is controlled by the sequencer 12.

Measurement is started by switching the sample injection valve 10 to the buffer side, closing the switch 11, and applying a high voltage E to the capillary tube.

At this time, at the same time, the data processor 13 starts using the signal from the sequencer 12 and collects the signal from the detector 6.

Through the operations described above, measurement is started with the sample sandwiched between the initial filling liquid and the buffer solution.

In this way, the measurement ends when a predetermined time elapses, but at the same time as the measurement ends, the switch 11
2, the high voltage power supply E is turned off, the data processor 13 is stopped, and the data processor 13 is opened and the data processing unit 13 is opened and the data processor 13 is stopped, and the data processing unit 13 is opened by the signal from The operation of the apparatus related to a series of measurements is completed. However, if it is necessary to wash out the substance remaining in the capillary tube 1 or to clean the inner surface, the following operation is performed after the measurement is completed. That is, the injection valve 10 is switched and connected to the initial filling liquid side, the solvent switching valve 19 is connected to the cleaning liquid side, and the waste liquid switching valve 4 is connected to the waste liquid +1W.
Switch to J and clean the inside of the capillary tube 1. After the cleaning is completed, connect one solvent switching valve to the initial filling liquid side of the cleaning liquid, fill the capillary tube 1 with the initial filling liquid, and proceed to the initial state of measurement! make it

On the other hand, FIG. 2 is an explanatory diagram of the configuration of another embodiment of the present invention,
In the figure, the same symbols as in FIG. 1 are used with the same meaning, and redundant explanation will be omitted here. In the embodiment having the configuration as shown in FIG.
Then, set the initial filling liquid to the initial filling liquid tank 17 and the lower MIaiii tank 3.

Thereafter, the sample injection valve 10 is switched and connected to the initial filling liquid side, and the inside of the capillary tube 1 is filled with the initial filling liquid. At this time, the measuring hole in the sample injection valve 10 is filled with the sample using the microsyringe 9 (or autosampler).

Switch the sample injection valve 10 to the sample injection side.

A fixed amount of the sample is injected into the capillary tube 1 by the hydraulic pressure from the constant water level generator 8. This injection amount is determined by the time that the measuring hole is connected to the inlet side of the capillary tube 1, and this time is controlled by the sequencer 12.

Set the sample injection valve 10 to Ra! ! Measurement is started by switching to the i il side, closing the switch 11, and applying the high voltage E. At this time, the data processor 13 is simultaneously started by a signal from the sequencer 12 and collects the signal from the detector 6.

Incidentally, by using a buffer solution corresponding to the leading solution and Cheelinda solution in isotaco instead of the initial filling solution and buffer solution in the above explanation, the capillary electrophoresis device of the present invention itself can be converted into an intaco (capillary ring 3IL electrophoresis device). It can be used as an electrophoresis device).

In addition, all operations such as switching valves and opening/closing switches explained above are performed based on signals from the sequencer 12, and the palpitation from sample injection to detection of separated components is performed in a constant temperature chamber 16 at a constant temperature. It will be done.

<Effects of the Invention> According to the present invention as described in detail above, the capillary can be used not only for ordinary capillary electrophoresis in an air phoresis apparatus, but also as a ++n-tube isokinetic electrophoresis apparatus. There are advantages. Furthermore, by housing the capillary tube, the microsample injection device, the detector for detecting the separated sample components, etc. in a constant temperature bath, the Joule heat generated by the application of the high voltage power can be effectively suppressed. The capillary tube section and the sample are always kept at a constant temperature. Further, the capillary is supplied with is by the sample injection device.
Since a small amount of sample is injected with good reproducibility and the timing of this sample injection and the application of the high-voltage power source to both ends of the capillary tube always match, it is possible to perform analysis with good reproducibility.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the configuration of an embodiment of the present invention, and FIG. 2 is an explanatory diagram of the configuration of the embodiment of the present invention. 1...Capillary tube, 3...-
M @> (M tank, 4...solvent switching valve. 5...electrode, 6...detector, 8...
... Constant water level generator, 9 ... Syringe, 10.
...Minor sample injection valve, 11...Switch, 12...Shield controller, 13...
・Data processing device, 14... Ammeter, 15...
... Recorder, 16 ... Constant temperature bath, 17 ...
...Initial filling liquid tank, 18...Cleaning liquid tank, 19...Waste liquid switching valve

Claims (1)

[Claims] (1) Connect the positive and negative sides of a DC high-voltage power source to both ends of a capillary tube filled with a buffer solution, and use electrokinetic phenomena to extract the components contained in the sample injected into the capillary tube. An analyzer that performs qualitative or quantitative analysis of sample components, which includes a means for supplying and circulating a buffer solution at a constant water level, and a solvent switching valve that switches between supplying either the initial filling solution or the washing solution. It has a micro sample injection device that can change the amount, and a waste liquid switching valve for discharging the liquid remaining in the capillary tube before or after the start of analysis, and the capillary tube, the micro sample injection device, and the solvent switching valve. A valve and a detector for detecting the separated sample components are housed in a constant temperature bath, and switch means for applying the high voltage power source to both ends of the capillary tube is provided, and the solvent switching valve is switched to Supplying the initial filling liquid or washing liquid to the micro sample injection device, and switching the waste liquid switching valve when the capillary tube is filled with the initial filling liquid or washing liquid by the micro sample injection device; A capillary electrophoresis apparatus characterized in that when a certain amount of sample is injected, the waste liquid switching valve and the valve of the microsample injection device are switched, and then sequence control is performed so that the switches are closed at a certain timing. . 2) Connect the positive and negative sides of a DC high-voltage power source to both ends of a capillary tube filled with a buffer solution, and use the electrokinetic phenomenon to transfer the sample injected into the capillary tube to the sample components. The analyzer performs the qualitative and quantitative determination of the sample components by separating the sample components, and the analyzer includes a means for supplying and circulating a buffer solution at a constant water level, a means for supplying an initial filling solution, and a micro-sample injection device capable of changing the internal volume. , the capillary tube, the microsample injection device, and a detector for detecting the separated sample components are housed in a constant temperature bath, and a switch means is provided for applying the high voltage to both ends of the capillary tube. and supplying the initial filling liquid to the micro sample injection device, and controlling the sequence so that the switch closes at a certain timing when a certain amount of sample is injected into the capillary tube by the micro sample injection device. A capillary electrophoresis device characterized by: