JPH06194339A - Sample injection method for capillary electrophoresis system - Google Patents

Abstract

PURPOSE:To allow an accurate injection quantity to be specified with an absolute injection quantity of sample by injecting a marker solution into a capillary and migrating the marker solution through the capillary until a detector detects the marker solution using a buffer solution under a predetermined pressure. CONSTITUTION:A capillary electrophoresis system comprises a capilarry 4 and a detector 6 disposed at a part thereof, a reservoir 16 containing an electrophresis buffer and to which the sample injection end of the capilary 4 is inserted, a reservoir 18 for containing the electrophoresis buffer and to which the other end of the capillary 4 is inserted, a high voltage power supply 12, and an automatic sampler 2. A detection signal is fed from the detector 6 to a data processing section 20 where the signal is subjected to waveform processing and upper peak position thereof is detected. The time required for the marker solution to arrive at the detector 6 is detected along with the holding time to be elapsed for each separated component before arriving at the detector 6. Output signal from the data processing section 20 is fed to a controller 22, e.g. a microcomputer, which controls injection of sample into the capillary and the analysis operation.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a capillary electrophoresis apparatus for separating and analyzing charged substances or neutral substances such as amino acids, proteins and nucleic acids, and more particularly to a method for injecting a sample solution into the capillaries.

[0002]

2. Description of the Related Art In a conventional method for injecting a sample in a capillary electrophoresis apparatus, a marker solution is moved to a detector in a buffer solution under the same conditions as the sample injection, and the time required for the movement is calculated. Calculate the injection amount per unit time from the inner diameter of the capillary and the length from the injection end to the detector, determine the injection time corresponding to the set sample injection amount, and inject the desired amount of sample into the capillary. (For example, Japanese Patent Laid-Open No. 4-9756)
(See Japanese Patent Publication).

[0003]

In the conventional sample injection method, if the viscosity of the buffer solution and the actual sample are about the same, an accurate sample injection amount can be specified by an absolute amount. When the viscosities of the two are significantly different, there is a problem that the injection rate changes as the volume occupied by the sample in the capillary changes (the sample amount changes), resulting in an error in the injection amount.

The present invention provides a sample injection method in a capillary electrophoresis apparatus capable of defining an accurate sample injection amount by an absolute amount even when the viscosity of a buffer solution and an actual sample greatly differ. It is an object.

[0005]

Means for Solving the Problems A capillary, a detector provided in a part of the capillary, both ends of the capillary are respectively inserted, and a migration voltage is applied to a reservoir containing a migration buffer and a sample injection side reservoir. In order to achieve the above object, in a capillary electrophoresis apparatus comprising a high-voltage power supply, in the sample injection method of the present invention, a marker solution is injected into a capillary, and the marker solution is a buffer solution under a predetermined pressure, and the marker solution is a detector. Is moved in the capillary until it is detected by, and the time tm required for moving the marker solution from the capillary injection end to the detector is measured,
Injection pressure P, marker solution movement time tm, capillary inner radius a, total length l, length from injection end to detector l _D
The viscosity of the buffer solution η ₁
Η ₁ = P · tm × a ² / 8l·l _D Again, the marker solution is moved in the tm ′ capillary for a certain time under a predetermined pressure with the buffer solution, and then the sample solution is injected to make the marker solution. Is moved in the capillary until the detector is detected, the marker solution movement time tm ″ after sample injection is measured, the marker solution movement measurement time tm ″ after sample injection, the injection pressure P, and the inner diameter of the capillary are measured. a ² , the total length l, the length l ″ of the sample injected into the capillary, and the viscosity η ₁ of the buffer solution, the viscosity η ₂ of the sample solution is calculated by the following equation, and (η ₂ −η ₁ ) l ” ² + 2l · η ₁ · l” = a ² · P ·
tm ″ / 4, l ″ = (tm−tm ′) × a ² · P / 8l ·
η ₁ .

Viscosity η _{2 of} sample solution and viscosity η _{1 of} buffer solution, inner diameter a ^{2 of} capillary, total length l, injection pressure P
Then, the injection time corresponding to the set sample injection amount is determined, and the sample solution is injected based on this.

[0007]

DESCRIPTION OF THE PREFERRED EMBODIMENTS A sample injection method in a capillary electrophoresis apparatus of the present invention will be described below with reference to the drawings. FIG. 1 is a schematic view of a capillary electrophoresis apparatus for carrying out the method of the present invention, and the capillary electrophoresis apparatus is a capillary. 4, a detector 6 provided on a part of the capillary 4,
A reservoir 16 for accommodating the migration buffer, into which the sample injection end of the capillary 4 into which the sample has been inserted is inserted, a reservoir 18 also containing the migration buffer and into which the other end of the capillary 4 is inserted, and a sample injection side reservoir 16 A high-voltage power supply 12 for applying an electrophoretic voltage, an autosampler 2 for injecting a sample into the capillary 4, and the like are provided.

The autosampler 2 automatically injects a sample into the capillary 4 by the injector 14, injects a marker solution for calibrating the injection amount, and further contains a migration buffer, a marker solution and a sample. The capillary 4 is moved between the respective containers 30, 34, 38 and the reservoir 16 (see FIG. 3). The sample and the like are injected by pressurizing a closed container with nitrogen gas or the like.

The capillary electrophoresis apparatus is placed in a constant temperature bath and its temperature is controlled in order to keep the temperature constant. In FIG. 1, the alternate long and short dash line indicates the temperature controlled range.

The detection signal from the detector 6 is the data processing unit 2
0 is input, and peak position detection is performed on waveform processing. Further, as will be described later, the time required for the marker solution to reach the detector 6 and the holding time for each separated component to reach the detector 6 in the analysis are detected. The signal from the data processing unit 20 is input to a controller 22 such as a microcomputer, and the capillary 4
Controls sample injection operation and analysis operation. Further, the controller 22 is preliminarily input with dimension setting values of the capillaries 4 to be described later, that is, information such as the inner diameter, the total length, the length from the injection end to the detector, and the like.

FIG. 2 shows an example of the detector 6. The measurement light 24 is emitted from one side of the capillary 4, and the transmitted light is detected by the photocell 28 arranged on the other side. 26
Is a slit arranged on the front surface of the photocell 28.

Hereinafter, the sample injection method of the present invention will be described with reference to FIGS. 3 and 4. As shown in FIG. 3A, the injection end of the capillary 4 is inserted into the container 30 containing the marker solution 32, and the sealed container is supplied with nitrogen gas at a constant pressure for a predetermined time to pressurize the marker solution 32. Inject into the capillary 4. The marker solution 32 may be anything as long as it responds to the detector 6 and is not adsorbed to the capillaries 4. For example, methanol, acetone or the like can be used. Further, it is desirable that the viscosity thereof is similar to that of the buffer solution.

Next, as shown in FIG. 3 (B), the capillary end into which the marker solution 32 has been injected is transferred to a container 34 containing an electrophoretic buffer solution 36, and the inside of the container 34 is pressurized with the same pressure as at the time of injection. Then, the marker solution 32 is extruded and moved in the capillary, and the marker solution zone 32 a is detected by the detector 6. In the data processing unit 20, the detector 6
The time tm required for the marker solution zone 32a to move from the capillary injection end to the detector 6 is measured on the basis of the signal from, and is taken into the controller 22. Information such as the pressure P at the time of injection and the dimension setting value of the capillary 4, that is, the inner diameter a ^{2 of} the capillary, the total length l, the length l _D from the injection end to the detector, etc. are input to the controller 22 as parameters. The viscosity η _{1 of the} buffer solution is obtained from each parameter of the measured marker solution movement time tm as follows.

A capillary of length l having an inner diameter a ² has a viscosity η
The flow rate V when filled with the solution of _{No. 1} and given a predetermined pressure is given by the Poiseville equation as follows.

V = πa ⁴ P / 8lη ₁ (1) Therefore, the average velocity v is v = a ² P / 8lη ₁ (2), and the length from the injection end to the detector is l _D. if the time required for this and _{tm, l D = vtm ...... (} 3) , and the formula (2) (3) from _{η 1 = P · tm × a} 2 / 8l · l D ...... (4) next The viscosity η _{1 of the} buffer solution can be calculated by substituting the above parameters into the equation (4).

Here, as shown in FIG. 4 (A), consider a case where a portion x of the entire length 1 of the capillary 4 is occupied by a solution of viscosity η ₂ . The average velocity Vx when pressurized with the same pressure as above is Vx = a ² P / 8 × 1 / (η ₂ −η ₁ ) x + lη ₁ (5) when this equation (2) is transformed, and this equation The value of (5) at x = 0 is given by the above equation (2).

Then, as shown in FIG. 4 (B), the end of the capillary into which the marker solution 32 has been injected is transferred to the container 34 containing the buffer solution 36 for migration in the same manner as described above, and the container has the same pressure as that at the time of injection. The inside of 34 is pressurized, the marker solution zone 32a is pushed out to move in the capillary, and the injection operation is interrupted at a time tm 'shorter than the time tm. The moving distance l'at this time is given by l '= vtm' by the equation (2), and the distance l "between the marker solution zone 32a and the detector 6 is l" = v (tm-tm '). .

Thereafter, as shown in FIG. 3 (D), the capillary end is transferred to the container 38 containing the sample 40, and the inside of the container 38 is pressurized again at the same pressure as that at the time of the injection, and the marker solution 3 is added.
2 and the buffer solution 36 are pushed out and moved in the capillary until the marker solution zone 32a is detected by the detector 6. At this time, the timer is reset and the time during which the sample is injected, that is, the marker solution movement time tm ″ after the sample injection is measured.

Therefore, as shown in FIG. 4C, time t
This means that the sample 40 is injected by the length of the distance l ″ during m ″.

When the above equation (5) is solved, (η ₂ −η ₁ ) x ² + 2l · η ₁ x = a ² · t / 4 (6), and t = tm ″, x = l ″ is substituted, the injection pressure P,
Since the parameters of the inner diameter a ^{2 of} the capillary, the total length 1 and the viscosity η ₁ of the buffer solution are known, the viscosity η _{2 of the} sample solution can be obtained from this.

As a result, the controller 22 determines from the parameters of the viscosity η _{2 of the} sample solution and the viscosity η _{1 of the} buffer solution, the inner diameter a ^{2 of} the capillary, the total length l, and the injection pressure P,
Using the above equation (6), an arbitrary x (0 <x <l), that is, the injection time t corresponding to the set sample injection amount is determined,
Based on this, the injector 14 performs the injection of the sample solution for the determined injection time.

After injecting the sample solution, the end of the capillary is shown in FIG.
As shown in (C), it is transferred to the reservoir 16 and a migration voltage is applied according to an instruction from the controller 22 to start the analysis.

[0023]

Since the present invention is configured as described above, there is a case where the relationship between the injection time and the injection amount is not linear because the viscosity of the buffer solution and the actual sample are significantly different. Also, the set arbitrary sample injection amount can be defined by an accurate absolute amount.

Further, the fact that the viscosity of the sample is greatly different from that of the buffer solution not only affects the injection amount, but also changes the overall viscosity, which affects the migration time at the time of analysis. However, since the viscosities of the buffer solution and the actual sample are known at the time of sample injection, it is possible to correct the migration time at the time of analysis by this.

[Brief description of drawings]

FIG. 1 is a schematic view showing an embodiment of an apparatus for carrying out the method of the present invention.

FIG. 2 is a diagram showing an example of a detector of an embodiment apparatus for carrying out the method of the present invention.

FIG. 3 is a diagram illustrating a method of the present invention.

FIG. 4 is a diagram illustrating a method of the present invention.

[Explanation of symbols]

2 ... Autosampler 4 ... Capillary 6 ... Detector 12 ... High-voltage power supply 16, 18 ... Reservoir 20 ... Data processing unit 22 ... Controller 30, 34, 38 ...
Container 32 ... Marker solution 36 ... Buffer solution 40 ... Sample

Claims (1)

[Claims] 1. A capillary, a detector provided on a part of the capillary, and both ends of the capillary are respectively inserted,
In a capillary electrophoresis apparatus consisting of a reservoir containing a migration buffer and a high-voltage power supply that applies a migration voltage to the sample injection side reservoir, inject the marker solution into the capillary and use the buffer solution under the prescribed pressure to prepare the marker solution. Is moved through the capillary until it is detected by the detector, and the time required for the marker solution to move from the capillary injection end to the detector is measured, and the injection pressure, marker solution transfer time, and capillary inner diameter, total length, injection Obtain the viscosity of the buffer solution from the length from the end to the detector, inject the marker solution into the capillary again, and move the buffer solution in the capillary for a certain time under a predetermined pressure,
Then, inject the sample solution and move it through the capillary until the marker solution is detected by the detector, measure the marker solution transfer time after sample injection, and then move the marker solution transfer time after sample injection, injection pressure, and capillary. The inner diameter, total length of the sample, the length of the sample injected into the capillary, and the viscosity of the buffer solution determine the viscosity of the sample solution.The sample injection is set from the viscosity of the sample solution and the buffer solution, the injection pressure, the inner diameter of the capillary, and the total length A sample injection method for a capillary electrophoresis apparatus, comprising determining an injection time corresponding to an amount and injecting a sample solution based on the injection time.