JP3186282B2 - Sample injection method for capillary electrophoresis device - Google Patents

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 or neutral substances such as amino acids, proteins and nucleic acids, and more particularly to a method for injecting a sample solution into a capillary in the apparatus.

[0002]

2. Description of the Related Art In a conventional sample injection method in a capillary electrophoresis apparatus, a marker solution is moved with a buffer solution through a capillary to a detector under the same conditions as in sample injection, and the time required for the movement is determined. 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, see Japanese Unexamined Patent Publication No.
Reference).

[0003]

In the conventional sample injection method, if the viscosity of the buffer solution and the actual sample are substantially the same, the accurate sample injection amount can be defined by the absolute amount. When the viscosities of the two are significantly different, the injection speed changes as the volume occupied by the sample in the capillary changes (sample amount changes), and there is a problem that errors occur in the injection amounts.

An object of the present invention is to provide a sample injection method in a capillary electrophoresis apparatus capable of specifying an accurate sample injection amount by an absolute amount even when the viscosity of a buffer solution and an actual sample are largely different. It is an object.

[0005]

Means for Solving the Problems A capillary, a detector provided in a part of the capillary, and both ends of the capillary are inserted respectively, 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, a capillary electrophoresis device comprising a high-voltage power supply and a sample solution of the present invention, wherein the marker solution is injected into the capillary, and the marker solution is detected with a buffer solution under a predetermined pressure. Is moved through the capillary until it is detected in the above, and the time tm required for the marker solution to move from the capillary injection end to the detector is measured.
Injection pressure P, marker solution transfer time tm and capillary inner radius a, total length l, length from injection end to detector l _D
From the following parameters, the viscosity η ₁
Look, eta ₁ = a ^{P · tm × a 2 / 8l} · l D again marker solution is moved for a predetermined time tm 'in the capillary under a predetermined pressure in the buffer solution, the marker solution is poured thereafter, the sample solution Is moved in the capillary until is detected by the detector, the marker solution movement time tm '' after the sample injection is measured, the marker solution movement measurement time tm '' after the sample injection, the injection pressure P, the inner diameter of the capillary 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 are used to determine the viscosity η ₂ of the sample solution according to the following equation, and (η ₂ −η ₁ ) l '' ² + 2l ・ η ₁・ l '' = a ²・ P ・
tm ″ / 4 where l ″ = (tm−tm ′) × a ² · P / 8l ·
It is η _1.

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
Then, the injection time corresponding to the set sample injection amount is determined, and the sample solution is injected based on this.

[0007]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view of a capillary electrophoresis apparatus for carrying out the method of the present invention; FIG. 4, a detector 6 provided in a part of the capillary 4,
A reservoir 16 containing the electrophoresis buffer and into which the sample injection end of the capillary 4 into which the sample has been injected is inserted, a reservoir 18 also containing the electrophoresis buffer and into which the other end of the capillary 4 is inserted, and a reservoir 16 on the sample injection side A high voltage power supply 12 for applying a migration voltage to the sampler, an autosampler 2 for injecting a sample into the capillary 4, and the like.

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 accommodates an electrophoresis 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). Injection of a sample or the like is performed by pressurizing a sealed container with nitrogen gas or the like.

[0009] The capillary electrophoresis apparatus is placed in a thermostat so as to keep the temperature constant, and the temperature is controlled. The dashed line in FIG. 1 indicates the range in which the temperature is controlled.

The detection signal from the detector 6 is transmitted to the data processing unit 2
0, and the peak position is detected in the waveform processing. Further, as described later, the time required for the marker solution to reach the detector 6 and the retention time during which the separated components reach the detector 6 are detected in the analysis. A signal from the data processing unit 20 is input to a controller 22 such as a microcomputer, for example, and the capillary 4
Controls the operation of injecting a sample into the device and the operation of analysis. In addition, information such as a dimension setting value of the capillary 4 described later, that is, information such as an inner diameter, a total length, and a length from the injection end to the detector is input to the controller 22 in advance.

FIG. 2 shows an example of the detector 6. The measurement light 24 is irradiated from one side of the capillary 4 and the transmitted light is detected by a 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. As shown in FIG. 3A, the injection end of the capillary 4 is inserted into the container 30 containing the marker solution 32, and a constant pressure of nitrogen gas is supplied to the sealed container for a predetermined time to pressurize the marker solution 32. Inject into capillary 4. As the marker solution 32, any solution that responds to the detector 6 and does not cause adsorption to the capillary 4 or the like may be used. For example, methanol, acetone, or the like can be used. Further, the viscosity is desirably the same as that of the buffer solution.

Next, as shown in FIG. 3B, the capillary end into which the marker solution 32 has been injected is transferred to a container 34 containing an electrophoresis buffer solution 36, and the inside of the container 34 is pressurized at the same pressure as at the time of injection. Then, the marker solution 32 is pushed out 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 32 a to move from the capillary injection end to the detector 6 is measured based on the signal from the controller 22 and is taken into the controller 22. Information such as the pressure P at the time of injection and the dimension set value of the capillary 4, that is, information such as the capillary inner diameter a ² , the total length l, and the length l _D from the injection end to the detector are input to the controller 22 as parameters. The viscosity η _{1 of the} buffer solution is obtained as follows from each parameter of the measured marker solution movement time tm.

A capillary having a length l of an inner diameter a ² has a viscosity η.
The flow rate V when filled with the solution ₁ and given a predetermined pressure is given by Poiseville's equation as follows.

V = πa ⁴ P / 8lη ₁ (1) Accordingly, the average velocity v is given by v = a ² P / 8lη ₁ (2), and the length from the injection end to the detector is _ID. If the time required for this is assumed to be tm, then l _D = vtm (3), and from equations (2) and (3), η ₁ = P · tm × a ² / 8l·l _D (4) By substituting the above parameters into Equation (4), the viscosity η _{1 of the} buffer solution can be calculated.

Here, as shown in FIG. 4A, a case where a solution having a viscosity η ₂ occupies a certain portion x of the entire length 1 of the capillary 4 will be considered. The average velocity Vx when pressurized at the same pressure as above is obtained by transforming the above equation (2) into the following equation: Vx = a ² P / 8 × 1 / (η ₂ −η ₁ ) x + 1η ₁ (5) The value at x = 0 in (5) is the above equation (2).

Next, as shown in FIG. 4B, the capillary end into which the marker solution 32 has been injected is transferred again to the container 34 containing the buffer solution for electrophoresis 36, and the container is kept at the same pressure as the injection. The inside of the capillary 34 is pressurized, the marker solution zone 32a is pushed out and moved 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 ′ according to 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. 3D, the end of the capillary is transferred to a container 38 containing the sample 40, and the inside of the container 38 is pressurized again at the same pressure as at the time of injection, so that the marker solution 3
2 and the buffer solution 36 are extruded and moved through 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.
This means that the sample 40 has been injected by the distance l ″ during m ″.

Here, when the above equation (5) is solved, (η ₂ −η ₁ ) x ² + 2l · η ₁ x = a ² · t / 4 (6) where t = tm ″ and x = l '', the injection pressure P,
Since the parameters of the capillary inner diameter a ² , the total length l, and the viscosity η ₁ of the buffer solution are known, the viscosity η _{2 of the} sample solution is obtained from this.

As a result, the controller 22 calculates 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 from the respective parameters.
Using the above equation (6), an arbitrary x (0 <x <l), that is, an injection time t corresponding to the set sample injection amount is determined,
Based on this, the injector 14 executes the injection of the sample solution for the determined injection time.

After injection of the sample solution, the capillary end is
As shown in (C), the sample is moved to the reservoir 16, the electrophoresis voltage is applied according to an instruction from the controller 22, and the analysis is started.

[0023]

Since the present invention is configured as described above, the relationship between the injection time and the injection amount is not linear due to the great difference between the viscosity of the buffer solution and the actual sample. Also, any set sample injection amount can be defined by an accurate absolute amount.

Further, the fact that the viscosity of the sample is significantly different from that of the buffer solution not only affects the injection volume but also changes the overall viscosity, which in turn affects the electrophoresis time during analysis. However, since the viscosity of the buffer solution and the actual sample is known at the time of sample injection, it is also possible to correct the migration time at the time of analysis.

[Brief description of the drawings]

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

FIG. 2 is a diagram illustrating an example of a detector of an embodiment apparatus for performing the method of the present invention.

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

FIG. 4 is a diagram illustrating the 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

──────────────────────────────────────────────────続 き Continued on front page (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 27/447 CA (STN)

Claims (1)

(57) [Claims] Claims: 1. A capillary, a detector provided in a part of the capillary, and both ends of the capillary are inserted, respectively.
In a capillary electrophoresis apparatus comprising a reservoir containing an electrophoresis buffer and a high-voltage power supply for applying an electrophoresis voltage to a sample injection side reservoir, a marker solution is injected into the capillary, and the buffer solution is applied under a predetermined pressure with a buffer solution. Is moved in the capillary until is detected by the detector, 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, capillary inner diameter, total length, and injection are measured. Determine the viscosity of the buffer solution from the length from the end to the detector, inject the marker solution into the capillary again, move the buffer solution with the buffer solution under a predetermined pressure for a certain period of time,
Thereafter, the sample solution is injected and moved in the capillary until the marker solution is detected by the detector, the marker solution moving time after the sample injection is measured, the marker solution moving time after the sample injection, the injection pressure, the capillary. Determine the viscosity of the sample solution from the internal diameter, full length of the sample, the length of the sample injected into the capillary, and the viscosity of the buffer solution, and inject the sample from the viscosity of the sample solution and buffer solution, the injection pressure, the internal diameter of the capillary, and the total length. A sample injection method for a capillary electrophoresis device, comprising: determining an injection time corresponding to an amount, and injecting a sample solution based on the determined injection time.