JPH0387648A - Capillary electro phoresis apparatus - Google Patents

Abstract

PURPOSE:To simultaneously measure samples having the polarities different in charge from each other as well, to improve reproducibility by providing a trace sample injector of variable capacities which can inject the samples into two capillary columns. CONSTITUTION:A constant water level forming device 8 which constitutes the trace sample injecting device is so provided that a buffer soln. is sent by the head difference generated between the capillary columns 1 and 1'. The buffer soln. stored in a buffer soln. tank 8d is thus fed into the columns by a pump 8c from the bottom of a pipe 8a. The sol. overflowing from the pipe 8a is returned to the tank 8d through the pipe 8b. An initial packing liquid is fed by an initial packing liquid pump 17 when the flow passage of an injection valve 10 is connected to the columns 1, 1'. A washing liquid is fed by a washing liquid pump 18 when a selector 19 is selected. The sample is supplied by a microsyringe 9 and the specified slight amt. of the sample is injected into the columns 1, 1' by the injection valve 10. The trace sample injecting device which can inject sample into the two columns in such a manner is provided and, therefore, the simultaneous measurement of the samples is possible with one time of the sample injection.

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Application Field> The present invention relates to a capillary electrophoresis device, and more specifically to an electrokinetic chromatography device.
netic chronatography, E
K C), capillary liquid chromatography using electroosmotic flow (capillary liquid chromatography)
id chronatography with ele
various types of capillary electrophoresis (capillary electrophoresis), including capillary isotachophoresis,
electrophoresis.

CE) This invention relates to a capillary electrophoresis device that is worried about its reproducibility and is capable of simultaneously measuring samples with different charge polarities (that is, different moving directions) in an analytical method.

<Conventional technology> Capillary electrophoresis, electrokinetic chromatography,
Both capillary liquid chromatography, which uses electroosmotic flow, and capillary isotachophoresis are types of electrophoretic analysis methods that perform separation analysis using differences in the electrical mobility of a sample under high electric field. However, capillary electrophoresis differs greatly from other electrophoresis methods in that separation is performed inside a so-called capillary column, which is a hollow tube with an inner diameter of several hundred μm or less. However, the equipment used for such electrophoresis (so-called electrophoresis equipment) usually consists of a hollow capillary column, a high voltage power supply, a detector, and a sample injector (method), and the basic configuration is almost the same. Therefore, it is possible that all of these measurement methods can be used properly.

By the way, in order to perform analysis with good reproducibility in the electrophoretic analysis method as described above, it is necessary to satisfy all of the following requirements (1) to (2). In other words, ■ a constant, extremely small amount of sample is always injected into the capillary column, ■ the time from sample injection to the start of analysis is controlled to be constant, and ■ the effects of Joule heat due to the application of high voltage are controlled. Make it smaller. ■ Avoid being affected by gas generated by electrolysis; ■ Ensure that the inner surface of the capillary column and titfA surface are not contaminated by samples, etc.

In addition, in order to obtain good reproducibility of analysis results, the sample injection method, which is one of the above requirements, is particularly problematic.

On the other hand, conventional sample injection methods include injection using the difference in the water level of the sample solution (spatula ζ difference), injection using electrophoresis and electroosmotic flow, and split injection. injection method (for example, Utility Model Application Publication No. 63-1)
61355), etc., but these sample injection methods have problems in terms of reproducibility of sample injection and ease of automation. As one method to solve this problem between automation and reproducibility, an injection method using a variable volume microvalve has been proposed (for example, in Japanese Patent Application Laid-Open No. 63-253
247), is said to be the best injection method at present because it is easy to automate and shows good reproducibility. However, in principle, these sample injection methods cannot be used for simultaneous measurement of minute amounts of samples using two columns, and due to problems with the valve mechanism when used for capillary isotachophoresis, It had the disadvantage that it could not be used for all of the analysis techniques mentioned above.

<Problems to be Solved by the Invention> The present invention has been made in view of the above situation, and the technical problem to be solved is to enable simultaneous measurement of samples having different polarities and to achieve reproducibility. Our objective is to provide an excellent capillary electrophoresis device.

Means for Solving the Problems> The features of the present invention that solve the above-mentioned technical problems include a means for supplying and circulating the Mfr liquid at a constant water level, an initial filling liquid and a washing liquid in a capillary electrophoresis apparatus. A variable volume micro sample injector which has a means for supplying a sample and a means for switching between an initial filling liquid and a washing liquid and is capable of injecting a sample into two capillary columns, and a residual liquid in the two capillary columns or It has a waste liquid switching valve for discharging the cleaning solution, and the two capillary columns, the micro sample injection device, the solvent switching valve, and the two detectors for detecting the separated sample components are placed in a constant temperature bath. and a switch means for applying the high voltage power source to both ends of the two capillary columns, and switching means for switching the waste liquid when the initial filling liquid or washing liquid is filled in the two capillary columns by the microsample injection device. In addition to switching the valve, when a certain amount of sample is injected by the sample injection device, samples with different charge polarities can be simultaneously measured by switching the waste liquid switching valve and the valve of the two-column microsample injection device. It's about doing.

Effect〉 The invention works as follows.

That is, a micro sample injection device with a variable capacity that can inject samples is connected to two capillary columns, a detector is provided on the downstream side of each of the two capillary columns, and the end of the capillary beyond that is filled with a buffer solution. M that was done
It is inserted into the buffer tank together with the electrode. The two electrodes in the two buffer tanks are each connected to a high-voltage DC power source so that positive and negative charges are applied to them.

Samples can be injected into the two capillary columns, which have two flow paths: one for supplying and circulating the M loading liquid at a constant water level (head pressure) and the other for supplying the solvent as the initial filling liquid and washing liquid. The sample to be measured is injected in extremely small amounts into the two capillaries by the variable-capacity microsample injection device. Here, when a high-voltage power source is applied to the two electrodes, the sample injected into each capillary column moves toward the electrode whose polarity is opposite to that of the sample.

In the capillary column inserted into the downstream 1lII slow-flowing liquid tank to which the negative electrode is connected, positively charged components of the injected sample move toward the downstream electrode and are detected by the detector. At this time, the sample with a negative charge moves in the opposite direction to the component with a positive charge, so it comes out of the capillary column into the flow path where the 67 liquid circulates, but because the flow rate of the circulating slow liquid is high, washed out of the sample injector. In the capillary column inserted into the downstream M buffer tank to which the positive electrode is connected, negatively charged components are similarly detected.

Furthermore, in this capillary electrophoresis device, the reproducibility of measurements is improved as follows. ■Since the sample injection device injects the sample using a buffer solution at a constant pressure,
The composition of the sample components hardly changes.

(2) By switching the channel for supplying the initial filling liquid to a cleaning liquid, dirt in the capillary column can be washed out, making it possible to perform measurements with good reproducibility.

At this time, in order to prevent contamination of the buffer solution by the cleaning solution from which dirt has been removed, the waste switching valve is provided to prevent the buffer solution from flowing into the lower buffer tank.

■To improve the reproducibility of measurements due to the generation of Joule heat, the capillary column, the microsample injection device, the waste liquid switching valve, and the detector for detecting the separated sample components were housed in a constant temperature bath. As a result, the Joule heat generated by the application of the high voltage is well dissipated, the capillary column part is always kept at a constant temperature, and the temperature of the injected sample is also kept constant, resulting in good measurement reproducibility. is obtained. In addition, eight detectors are placed inside the thermostatic chamber, making them unaffected by the external environment and allowing highly sensitive measurements.

■Injection of the initial filling liquid, injection of the sample, application of high voltage, cleaning of the capillary column, and switching of the two switching valves mentioned above are all controlled by the sequencer, allowing for highly reproducible injection and measurement.

■The micro sample injection device with a variable capacity that can inject samples into the two capillary columns has a flow path that supplies and circulates a buffer solution at a constant water level (head pressure), and a solvent that serves as an initial filling solution and a washing solution. Since it has two channels, one for the measurement of isotachos, it can be used for both normal capillary electrophoresis and isotachometry.

<Example> Hereinafter, the present invention will be explained in detail using the drawings. 1st
The figure is a configuration explanatory diagram of an embodiment of the present invention.

In this figure, 8 is a constant water level generator, and the buffer solution (solvent A) is fed by the head difference generated between the capillary columns 1 and 1''. The two wooden pipes 8a and 8b are connected in the middle,
The M buffer tank 8d is pumped from the bottom of the pipe 8a by the pump 8C.
The WI liquid stored in is sent. The solvent that overflowed in the tube 8a passes through the tube 8b and returns to the buffer tank 8d.
is being refluxed to.

Reference numeral 17 denotes an initial filling liquid pump, which pumps the initial filling liquid (solvent B) when the flow path in the sample injection valve 10 is connected to the capillary columns 1 and 1'. In addition, when flushing the cleaning liquid, switch one switching valve and
The washing liquid pump is used to send the washing liquid to the capillary columns 1 and 1'.

9 is a microsyringe for supplying a sample, and 10 is a sample injection valve for injecting a predetermined minute amount of sample into the capillary column 1.

4.4' is a waste liquid switching valve, which connects the capillary columns 1,1' to the ribbon wI liquid tank 3'3' during measurement, but when the initial filling liquid or washing liquid flows through the capillary column 1,1'. is switched to the waste liquid side.

Reference numeral 11 denotes a switch for applying a high voltage power supply E to the capillary columns 1, 1', and is operated by a control signal from a sequencer to be described later.

A sequencer 12 provides control signals to the sample injection valve 10, the switch 11, and the signal processing section 13 according to a predetermined sequence. Note that 14 is an ammeter and 15 is a recorder.

16 is a constant temperature bath, inside of which a sample injection valve 10, a capillary column 1.1', a detector 6.6', a switching valve 4.4', ! New liquid tanks 3, 3' are installed.

The operation of the embodiment of the present invention having the above-mentioned configuration will be explained in detail below. First, set the target II liquid A in the net WI liquid tank 8d of the constant water level generator 8, operate the circulation pump 10c, and fill the buffer tank 10a with buffer solution A. Next, add the target buffer B to the initial stage. Filling liquid pump 1
7 and lower buffer tank 3.3'. If it is necessary to wash the inside of the capillary column 1.1, a washing liquid is set in the washing liquid pump 18.

Next, 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°4' is connected to the waste liquid side, and the inside of the capillary column 11° is connected. Fill with initial filling liquid. At this time, use a microsyringe or an autosampler to insert the sample into the measuring holes of the sample injection valve <103b and 10.
4b).

After that, switch the sample injection valve 10 to the sample injection side,
Switch the waste liquid switching valve 4.4'' to the buffer tank 3, 3' side. A fixed amount of the sample is injected into the capillary column 1 by the liquid pressure from the constant water level generator 8. 104b) is connected to the entrance side of the capillary column 1.1゛,
This time is controlled by the sequencer 12.

Next, the sample injection valve 10 is switched to the yI solution, the switch 11 is closed, and the high voltage power source E is applied to start measurement. At this time, the data processor 13 starts at the same time with a signal from the sequencer 12, and the detector 6.6
Collect signals from °. At this time, the measurement is started with the sample being sandwiched between the initial filling solution and the buffer solution.

Thereafter, the measurement ends when a predetermined time has elapsed, but at the same time as the measurement ends, the switch 11 is opened by a signal from the sequencer 12, the high voltage power supply E is cut off, and the data processor 13 is stopped. Obtained data (
Performs computational analysis of talomadograms, intercopherograms, etc.

In this way, the operation of the device related to a series of measurements is completed, but if it is necessary to wash out the residue inside the capillary column 1.1° or to clean the inner surface, perform the following operation after the measurement is completed: Inject the sample. The valve 10 is switched and connected to the initial filling liquid side, the solvent switching valve 19 is connected to the washing liquid state, and the waste liquid switching valve 4.4° is switched to the waste liquid side to wash the inside of the capillary columns 1, 1'. After the washing is completed, one solvent switching valve is connected between the washing liquid and the initial filling liquid, and the inside of the capillary column 11 is filled with the initial filling liquid to set the initial state for measurement. In addition, by using Mice, which corresponds to the leading liquid and the melting liquid in a capillary isotachophoresis device, instead of the initial filling solution and buffer solution described above, the capillary electrophoresis device of the present invention can be converted into a capillary electrophoresis device of the capillary type. Can be used as an isotachophoresis 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 operations from sample injection to detection of separated components are performed in a constant temperature chamber 16 at a constant temperature. It will be done.

On the other hand, FIG. 2 is an exploded perspective view of the sample injection valve, in which 101.102 is a disk-shaped valve seat, 1.03.1
04 is a disk-shaped rotor made of ceramic, for example, which is sandwiched between these valve seats, and the two disks rotate at the same time. A metering hole is formed in this pair of rotors by overlapping two disks. There is a hole I in the valve seat 101.
ota to 101d are provided, and the rotor 103 has a hole 1.
03a to 103e are provided, and the rotor 104 has a hole 1.
04, a to 104f are provided, and the valve seat 102 is provided with a hole 1.
02a to 102e are provided.

Shafts are inserted through the holes Iota, 103a, 104a, and 102a. In the hole 103e of the rotor 103, an arc-shaped groove 103f is formed on the surface in contact with the valve seat 101, and in the rotor 104, two large and small arc-shaped grooves 104g and 104h are formed in the surface in contact with the rotor 103. ,
An arcuate groove 1041 is formed on the surface in contact with the valve seat 102.

Among these holes, the hole 102e is an inlet for the buffer solution, and the hole 101 (I
is an MI bombardment liquid discharge port, hole 102C is an introduction port for initial filling liquid, hole 101b is a sample injection hole, and hole 102b is a sample discharge hole. Also, hole 101C.

102d is a hole to which a capillary column is connected.

FIG. 3 is an explanatory diagram for explaining the operation of the sample injection valve configured as described above.
′ and shows the state when filling the measuring hole with the sample.
Figure (c) shows the state at the time of sample injection. The operation of the sample injection valve when the sample injection device is used in a capillary type isotachophoresis apparatus will be described below with reference to FIG.

In the state before sample injection, that is, in the state filled with the initial filling liquid, the holes 101C11 and 03d are
, 104e, 102d are connected in a straight line, and the water 104
It is connected to the hole 102C, which is the introduction port for the initial filling liquid, through h. In addition, holes 101b, 103b, 104b, 1
02b is also connected in a straight line. In this state, the initial filling liquid from the initial filling liquid pump 17 flows into the capillary column, and the sample applied from the microsyringe 9 passes through the hole 101b and fills the measuring holes 103b and 104b. Excess sample is discharged from hole 102b.

When the rotors 103 and 104 are switched as shown in Figure (c), the metering hole (between 103b and 104b) is set to hole 1. OIC
, 102d to the capillary columns 1 and 1', and the sample filled in the measuring holes 103b and 104b flows through the capillary columns 1 and 1' as shown in the figure.
It is pumped within 1゛. Incidentally, the rotors 103 and 104 are switched at a constant timing by a motor or the like (not shown) based on a control signal fJjJ from the sequencer 12. A part of the sample filled in the measuring holes (between 103b and 104b) is injected into the JIr filter column 1.1';
b is connected to the capillary column 1.1° 1. Determined in relation to buffer head pressure.

After this, the rotors 103 and 104 are brought into the state shown in Figure (A). At this time, hole 102e, which is the introduction port for the M new solution, and hole 1
04f, 103e, hole 101 which is the outlet for the MW liquid
d are connected by grooves 104i and 103f. In this state, the buffer solution from the constant water level creator 8 is flowing as if washing the inlet of the capillary column 1.1'. Here, by opening the bottom of the switch 11, a high voltage power source E is applied to both ends of the capillary column 1.about.1', and separation is performed. After the measurement is completed, the switch 11 is turned off to complete the analysis.

Incidentally, since the sample injected into the capillary columns 1, 1' is a very small amount (1 to 1 onl), in the above embodiment of the present invention, a part of the sample filled in the measuring holes 1031) + 104b is injected partially. The above-mentioned minute amount of sample is obtained. However, the rotor 103.1.04 is made thinner and the diameters of the measuring holes 103b and 104b are made smaller so that the optimal amount of sample required for analysis can be obtained from the measuring holes 103b and 104b. If so, the entire amount of the sample filled in the measuring holes 103b, 104b is injected into the capillary columns 1, 1'.

<Effects of the Invention> According to the present invention as described in detail above, it has two capillary columns and two detectors, a means for supplying and circulating a buffer solution at a constant water level (head pressure), an initial filling liquid, By installing a variable volume micro sample injection device that can inject samples into two capillary columns, it is equipped with a means for supplying a solvent as a washing liquid and a means for switching between an initial filling liquid and a washing liquid. It becomes possible to measure simultaneously.

In addition, this microsample injection device has a means for supplying a solvent as an initial filling liquid and a washing liquid, and supplies the initial filling liquid into the capillary column by using a waste liquid switching valve and an initial filling liquid pump together. It can be used not only for ordinary capillary electrophoresis but also for capillary isotachophoresis. Further, by switching the switching valves for the initial filling liquid and the cleaning liquid, the cleaning liquid is sent instead of the initial filling liquid, thereby making it possible to wash out dirt in the capillary column and perform measurements with good reproducibility. can.

Measures to improve the reproducibility of measurements include the capillary column, the micro sample injection device, the waste liquid switching valve,
By housing the detector and the detector in a constant temperature bath, Joule heat is well dissipated, the capillary column and the sample are always kept at a constant temperature, and good reproducibility can be obtained.

Furthermore, since the timing of sample injection by the sample injection device and application of the high-voltage power source to both ends of the capillary column is controlled by a sequencer, analysis can be performed with high reproducibility.

Furthermore, in the capillary electrophoresis device of the embodiment of the present invention, when simultaneous analysis using two columns is not performed, by providing a blind plug to the connection point (101c) of one capillary column of the sample injection device, it is possible to It also has the advantage of being able to carry out measurements similar to those using an electrophoresis device.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram of the configuration of an embodiment of the present invention, FIG. 2 is an exploded perspective view of a micro-sample injection valve, and FIG. 3 is an explanatory diagram of the operation of the micro-sample injection valve. 11- Capillary column, 3...
・M new liquid tank, 4, 4'... Waste liquid switching valve, 8... Constant water level creator, 9...
Microsyringe, 10...Sample injection valve,
11... Switch, 12... Sequencer, 14... Ammeter, 15... Recorder, 16... Constant temperature chamber, 17. ...Initial filling liquid pump, 18...Cleaning liquid pump, (a) Figure 3 (b) (c)

Claims (1)

[Claims] The positive and negative sides of a DC high-voltage power supply are connected to both ends of a capillary column filled with a buffer solution, respectively, and components contained in a sample injected into the capillary column are collected using electrokinetic phenomena. In a capillary electrophoresis analyzer that performs qualitative and quantitative analysis of the sample components by separating them, the device 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 washing solution, and a means for supplying an initial filling solution and a washing solution. a variable-volume microsample injector having switching means and capable of injecting samples into two capillary columns; a waste liquid switching valve for discharging residual liquid or washing liquid from the two capillary columns before or after the start of analysis; comprising two capillary columns, the microsample injection device, the solvent switching valve, two detectors for detecting the separated sample components, and a switch means for applying the high voltage power source to both ends of the two capillary columns, When the initial filling liquid or washing liquid is filled into the two capillary columns by the micro sample injection device, the waste liquid switching valve is switched, and when a certain amount of sample is injected by the sample injection device, the waste liquid switching valve is switched. and a capillary electrophoresis device, characterized in that samples having different polarities of charge are simultaneously measured by switching the valves of the two-column microsample injection device.