JPS6258461B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an electrophoresis analyzer, and more particularly, to an electrophoresis analyzer that can easily perform a type of zone electrophoresis in addition to its original function of isokinetic electrophoresis. Regarding.

In isotachophoresis, two types of electrolytes, a terminal liquid and a leading liquid, are filled inside the electrophoresis tube, and substances that become charged at the interface between these two electrolytes, such as amino acids, peptides, biological substances, etc. A sample is injected and subjected to constant current electrophoresis, and the target substance is separated into a single compartment based on the difference in mobility, and qualitatively and/or quantitatively determined using a detector as appropriate. It has become indispensable for analysis.

However, for example, when the sample is in an extremely small amount or contains two components with extremely close mobilities, it is not possible to conduct a sufficient analysis using normal isotachophoresis. That is, in the former case, the width of the ionic component compartment of the obtained sample is too narrow for sufficient detection, and in the latter case, the ionic component compartments of the two types of components are too close to each other to distinguish them. There was something I couldn't do.

The present invention provides an improved electrophoresis analyzer that can suitably perform analysis even in the above-mentioned cases. In an electrophoresis analyzer in which a sample injection section and a detector are connected in order through a pipe between the sample injection section and the leading liquid electrode tank, a predetermined position of the pipe between the sample injection section and the detector is connected to the border of the ion component compartment. An electrophoretic analyzer is provided, which includes passage detection means for detecting passage of a surface, a branch pipe is connected to a pipe line near a predetermined position of the passage detection means, and an electrolyte injection means is connected to the branch pipe.

The above-mentioned predetermined position is not particularly limited as long as it is a position from the sample injection part to the detector side by an electrophoresis distance at which the ionic components of the sample are appropriately separated into sections by isokinetic electrophoresis.

Specific examples of the passage detection means include an ultraviolet absorbance detector, a potential gradient detector, and a conductivity detector, which are attached to the predetermined position of the conduit. In addition, a sudden change in pipe diameter is formed at the predetermined position of the pipe, and a voltage change measuring means for measuring temporal changes in the supply voltage is connected to the power supply circuit, so that the boundary surface of the ionic component section is located at the pipe diameter. One example is one in which passage is detected based on a peculiar change in the output of the voltage changing means that occurs when passing through a sudden change section. Here, a sudden change in pipe diameter refers to a constriction where the pipe diameter suddenly decreases or a bulge where the pipe diameter increases, and the pipe between the sample injection part and the detector is a pretube with a large pipe diameter. In the case of a two-stage tube in which a tube and a capillary reach tube having a narrow pipe diameter are connected in series via a stepped portion, this is the stepped portion. In addition, the voltage change measuring means is
For example, it is composed of an A-D converter and a microcomputer. In this case, for example, the microcomputer measures the voltage between both ends of the power supply circuit via the A-D converter at predetermined short intervals, and The difference between the measured value and the measured value is obtained as the voltage change.

The branch pipe is connected to a conduit in the vicinity of a predetermined position between the predetermined position and the sample injection section, or to a conduit in the vicinity of a predetermined position between the predetermined position and the detector.

The electrolytic solution injection means is, for example, a plunger pump, and manually or automatically injects the electrolytic solution into the conduit via the branch pipe based on the output signal of the passage detection means. Depending on the timing of injection, the electrolyte can be injected arbitrarily before or after the target ion component compartment of the sample, but if it is injected before, the electrolyte should have a lower mobility than the target ion component compartment, and if it is injected later, the electrolyte should have a lower mobility than the target ion component compartment. Generally, an electrolyte with higher mobility is used.

For example, if electrophoresis is continued by injecting a liquid of the same type as the leading solution as an electrolytic solution with high mobility after the target ion component compartments of the sample, the target ion component compartments will substantially move through the leading solution. Therefore, the same effect as zone electrophoresis can be obtained.

Therefore, in the electrophoresis analyzer of this invention, isokinetic electrophoresis and a type of zone electrophoresis are performed continuously, and samples that cannot be sufficiently analyzed by normal isokinetic electrophoresis alone can be suitably analyzed. You will be able to do it.

Hereinafter, the present invention will be explained in more detail based on embodiments shown in the drawings.

Reference numeral 1 shown in FIG. 1 is an embodiment of an electrophoretic analyzer according to the present invention. A terminal liquid electrode tank 3 and a leading liquid electrode tank 4 are connected to both ends of the constant current power supply circuit 2, and a sample injection part 5 is connected between these.
and a detector 6 are connected through a conduit 7 to constitute a basic analysis section. Sample injection section 5 and detector 6
The pipe line 7 between the two is a pretube 7a with a large pipe diameter.
This is a two-stage tube in which a capillary reach tube 7b having a narrow pipe diameter and a capillary reach tube 7b are connected in series via a stepped portion 8. Detector 6 is an ultraviolet absorbance detector.

The stepped portion 8, which is a sudden change in pipe diameter, and the A-D converter 10 and microcomputer 11, which are voltage change measuring means, detect the passage of the boundary surface of the ionic component section through the stepped portion 8. constitutes a detection means. That is, A-D converter 1
0 converts the voltage supplied from the power supply circuit 2 into a digital quantity and outputs it to the microcomputer 11, and the microcomputer 11 converts the A-D converter 10 at predetermined short intervals based on the built-in clock.
, and subtract the value from the previous sampling from that value. The resulting difference value is the time variation of the supply voltage. When one ionic component compartment passes through the stepped portion 8, this time change is a steady change, but when the boundary surface of that ionic component compartment passes, other ionic component compartments with different conductivities change over time. This change is unique because it is adjacent to . Therefore, passage through the boundary surface can be detected.

12 is a branch pipe, which connects to the stepped portion 8 via a valve 13.
One end is connected to a conduit 7 slightly closer to the sample injection section 5 . Valve 13 is controlled by microcomputer 11.

Plunger pump 14 as electrolyte injection means
is connected to the other end of the branch pipe 12, and is used to inject a liquid of the same type as the leading liquid as an electrolyte into the pipe line 7 under the control of the microcomputer 11.

Next, I will explain the operation, but for the sake of explanation,
It is assumed that the sample consists of two types of ionic components, and these components have extremely small amounts and have similar mobilities.

When isokinetic electrophoresis is first performed according to a known procedure, the sample undergoes electrophoresis while being separated into two adjacent ionic component compartments S ₁ and S ₂ as shown in FIGS. 3a and 3b. If uniform electrophoresis continues as it is, the temporal change in the voltage supplied from the power supply circuit 2, that is, the first derivative V', will be approximately as shown in FIG. 2. Here, at times t ₀ and t ₁ when the first-order differential V' changes rapidly, the rear end boundary surface of the ion component section L of the leading liquid and the front end boundary surface of the ion component section T of the terminal liquid cross the stepped portion 8, respectively. This is the time when the line passed, and these are detected by the microcomputer 11.

FIG. 3a schematically shows the state inside the pipe line before and after the stepped portion 8 at time _t0 , and FIG. 3b shows the state at time _t1 .

When the microcomputer 11 detects a peculiar change in voltage at time t ₁ , it opens the valve 13 , which had been closed until then, and operates the plunger pump 14 to inject the electrolyte L ₀ into the conduit 7 . This state is shown in FIG. 3c. When the injection is finished, the valve 13 is closed again.

Then, when electrophoresis is performed by further applying current, the ions of the injected electrolytic solution L ₀ have a high mobility like the leading solution L, so the ion component section S ₁ of the sample that had progressed before that has a high mobility. , S ₂ and some of the terminal fluid ionic component section T, and overtake them while overlapping.

At this time, the ionic component sections S ₁ and S ₂ of the sample and the ionic component section T of the terminal liquid undergo a type of zone electrophoresis, so the width of the sections is expanded as shown in Figure 3d. At the same time, the compartments are separated from each other by the ionic component compartment L ₁ of the electrolytic solution L ₀ .

Therefore, the data obtained by the detector 6 is
As shown in FIG. 4, the ionic component section S ₁ of the sample,
The widths of the peaks P ¹ and P ₂ corresponding to S ₂ are appropriately wide, and both peaks are clearly separated, making it suitable for analysis.

In contrast, data obtained only by normal isotachophoresis shows peaks as shown in Figure 5.
The widths of P ₁ and P ₂ are too narrow, and both peaks appear to be fused together, making it difficult to perform a thorough analysis.

As is clear from the above description, according to the electrophoresis analyzer of the present invention, it goes without saying that normal isokinetic electrophoresis can be performed, and in addition to isokinetic electrophoresis, a type of zone electrophoresis can be performed continuously. This can be done easily and at the right timing. Therefore, it is extremely useful for analyzing samples containing extremely small amounts of components.

[Brief explanation of the drawing]

Fig. 1 is an explanatory diagram of the configuration of an embodiment of the electrophoresis analyzer of the present invention, and Fig. 2 is a diagram of temporal changes in the first derivative of the supply voltage when performing isokinetic electrophoresis with the apparatus of Fig. 1. , Fig. 3 is an explanatory diagram of the progress of electrophoresis when electrophoresis is performed using the apparatus shown in Fig. 1, Fig. 4 is an illustration of data obtained by the apparatus shown in Fig. 1, and Fig. 5 is an illustration of the progress of electrophoresis when electrophoresis is performed using the apparatus shown in Fig. 1. It is a diagram of data obtained by electrophoresis. 1... Electrophoresis analyzer, 2... Constant current power supply circuit,
3...Terminal liquid electrode tank, 4...Leading liquid electrode tank, 5...Sample injection part, 6...Detector, 7...Pipe line,
8... Stepped portion, 10... A-D converter, 11... Microcomputer, 12... Branch pipe, 13... Valve, 14... Plunger pump, L... Ionic component compartment of leading liquid, L ₀ ... Electrolyte solution to be injected, L ₁
...Ionic component compartment by the injected electrolyte, T...Ionic component compartment of the terminal liquid, _S1 , _S2 ...Ionic component compartment of the sample.

Claims (1)

[Scope of Claims] 1. An electrophoresis analyzer in which a sample injection part and a detector are sequentially connected by a pipe between a terminal liquid electrode tank and a leading liquid electrode tank, which are respectively connected to both ends of a constant current power supply circuit. In this method, a passage detection means is provided for detecting that the boundary surface of the ion component compartment passes through a predetermined position of the conduit between the sample injection part and the detector, and a branch pipe is connected to the conduit near the predetermined position. , and an electrophoretic analyzer characterized in that an electrolytic solution injection means is connected to the branch pipe. 2. The electrophoresis analyzer according to claim 1, wherein the passage detection means is an absorbance detector installed at a predetermined position in the conduit. 3. The electricity according to claim 1, wherein the passage detection means comprises a pipe diameter sudden change part formed at a predetermined position of the pipe and a voltage change measuring means connected to a power supply circuit to measure temporal changes in the supply voltage. Migration analyzer. 4. The electrophoretic analyzer according to claim 3, wherein the sudden change in pipe diameter is a constriction where the pipe diameter becomes smaller at that position. 5. The electrophoretic analyzer according to claim 3, wherein the sudden change in pipe diameter is a bulge in which the pipe diameter increases at that position. 6 The pipe line between the sample injection part and the detector is a two-stage tube in which a pretube with a large pipe diameter and a capillary reach tube with a small pipe diameter are connected in series via a stepped part, and the pipe diameter suddenly changes. 4. The electrophoretic analysis device according to claim 3, wherein the stepped portion is the stepped portion. 7. Claim 1, wherein the branch pipe is connected to a conduit near the predetermined position between the predetermined position and the sample injection part, and the electrolyte to be injected is the same type of liquid as the leading liquid.
The electrophoretic analyzer according to any one of Items 1 to 6.