JPS5857703B2 - Preparative separation method in electrophoretic analysis - Google Patents

Description

[Detailed description of the invention] The present invention relates to a fractionation method in electrophoretic analysis.

More specifically, the present invention uses a detector placed at a predetermined position in a migration tube to detect a zone of single component ions migrating while being separated in the migration tube by electrophoresis, and based on this detection signal, The electrophoresis is stopped by opening the high-voltage power circuit that supplies a constant current to the leading and terminal electrolyte tanks attached to both ends of the electrolyte tank, and the electrophoresis is stopped within a predetermined time after stopping, and the detection zone is moved to the stopped position in the electrophoresis tube. The present invention relates to a preparative separation method in electrophoretic analysis, characterized in that a predetermined amount of a target component substance is separated by operating a preparative separation mechanism provided in an electrophoretic analysis.

In capillary electrophoresis, a capillary tube is filled with a terminal electrolyte and a leading electrolyte, and samples of substances (amino acids, peptides, biological substances, etc.) that become charged at the interface are collected. The target substance is separated (or fractionated) by electrophoresis using a constant current, and then qualitatively and/or quantitatively determined.

By the way, since the quality is determined by comparison with a standard product, it cannot be determined if there is no standard product.

In such cases, the zone in which the analyte has been separated (target zone) is usually sampled, and qualitative (or quantitative) analysis is performed separately.

Examples of specific mechanisms for performing such fractionation include:
One example is one in which a septum is provided in the electrophoresis tube and the fraction is collected using a microsyringe through the septum.

On the other hand, the above-mentioned fractionation has been proposed and carried out in a state where an electrophoresis current is applied.

This is because it was thought that after the current was turned off, there was a risk of mixing of the separation zones due to diffusion or the like.

Additionally, this type of electrophoresis device requires extremely high voltage (
For example, since (1) is supplied to No. 10, large-scale insulation measures must be taken during preparative separation to prevent danger and protect the equipment.

In view of these circumstances, the inventors conducted intensive research and discovered that in capillary electrophoresis analysis, if fractionation is performed within a certain period of time after the electrophoresis current is turned off, the influence of diffusion will not be so great. Based on this new knowledge, we have developed a new preparative method for electrophoretic analysis that is safe and easy to operate, as well as preventing diffusion and eliminating the need for insulation measures for the preparative separation mechanism. This is what I came up with.

In other words, one of the main features of the preparative separation method in electrophoretic analysis according to the present invention is that the preparative separation is performed without applying a high voltage, that is, by stopping the electrophoresis. The first feature is that the migration tube is bent in the middle, a detector and a septum are installed at specific positions corresponding to the bend, and a preparator passage is provided in the main body case corresponding to the septum. Another feature is that the high-voltage power supply switch is turned off by opening the separator passages.

Due to these features, the present invention concretely guarantees safety by not allowing preparative separation operations using a preparator to be performed without turning off the high-voltage power supply, and also enables straight-line reciprocating movement that is easy to automate. make it possible to do so.

The preparative separation method of the present invention will be described in detail below based on an example of an analyzer shown in the drawings.

Note that this invention is not limited to this.

First, in FIG. 1, a capillary type isotachophoresis analyzer 1 consists of a terminal liquid electrode tank 2, a capillary tube 5 in which a sample injection port 3 and a separation cell 4 are installed, a leading liquid electrode tank 6, and It mainly consists of a constant current high voltage power supply circuit 7 for both electrodes of the electrode tanks 2 and 6, and a main body case 8.

In FIG. 2, the preparative cell 4 is interposed in the middle of the capillary tube 5 to form a bent part 9 in the migration flow path, and a detector 10 is provided in front of the bent part, and a detector 10 is provided at the front stage of the bent part. each includes a septum 11 and a needle guide 12.

In other words, the bending portion 9 is configured to bend the migration channel at a substantially right angle, and the septum 11 and needle guide 12 are arranged coaxially with the preceding channel.

The detector 10 is mainly composed of two potential gradient electrodes 13 and 14 arranged at a predetermined interval, and can detect each separated substance and measure its moving speed.

The high voltage power supply circuit 7 includes a DC high voltage power supply 15 and a switch 16.
and a relay 17, and when the switch 16 opens and closes, the DC high voltage power supply 15 can be opened and closed through the relay 17.

Note that 18 is an operation switch for the DC high voltage power supply 15.

The main body case 8 has a passage side door 1 for the microsyringe M.
9, and this passage-side door is pivotally supported on a shaft 20 so as to be freely openable and closable so that the opening operation thereof causes the switch 16 to be opened.

Next, the operation of the capillary type isokinetic electrical turbulence analyzer 1 having the above-mentioned structure without using a support will be explained, and the fractionation method will be explained in detail.

First, in the sample injection port 3 of the capillary tube 5, align the boundary between an electrolyte containing anions with higher mobility than the sample (leading electrolyte) and an electrolyte containing anions with lower mobility (terminal electrolyte). A sample is injected in the direction of the boundary, and a constant current is supplied from the constant current high voltage power supply 15 to perform isokinetic electrophoresis.

In this way, the sample ions (anions) are separated (fractionated) into zones (bands) of middle component ions inside the electrophoresis tube in order of their mobility, and each zone is separated from each other while maintaining a clear boundary surface. It starts moving in the direction of arrow A at a constant speed with a certain width determined by the amount of ions.

In this case, a unique potential gradient is formed in each zone depending on its mobility, and this potential gradient is detected by the detector 1.
It is possible to know the single component ion detected and separated by 0.

That is, target substance ions to be separated can be detected from the potential gradient value.

When the front end boundary surface of the target substance zone to be separated is detected in this way, the detection signal is displayed by an appropriate means, and based on the display, the passage side door 19 of the main body case 8 is opened, and the opening port is opened. The preparative microsyringe M is moved in the direction of the arrow B through the needle M, and the needle m is inserted into the capillary tube 5 through the needle guide 12 and the septum 11.

That is, the needle m is inserted coaxially in the direction of arrow B until the front end of the cylinder C of the microsyringe M comes into contact with the needle guide 12, and eventually the tip of the needle m is held at a preset dispensing position P and reaches that position. A certain amount of the target substance is aspirated into the cylinder C, that is, aliquoted.

By the way, when the door 19 of the main body case 8 is opened, the switch 16 is operated to open, which causes the relay 17 to operate and the high voltage power supply circuit 7 to open.

Therefore, when the microsyringe M comes into contact with the preparative cell 4, the high voltage power supply circuit 7 is open, allowing safe operation.

As described above, the passage door 19 of the preparative cell 4 and the main body case 8
and the operation for sorting the sorting mechanism mainly composed of the microsyringe M, that is, the opening of the passage 7j19, the movement of the microsyringe M in the direction of arrow B, the suction, and the backward movement linked to the opening of the passage group 19. This turns off the high voltage power supply circuit.

Of course, this movement of the microsyringe may be performed automatically.

Next, we will give an example of an actual journey, and show that in preparative separation using the capillary isotachophoresis device 1 shown in Figures 1 and 2, the influence of separation zone diffusion is small within a certain time after the electrophoresis current is turned off. shows.

Experimental Example First, 1QCC of a mixed water bath solution containing 29.4 mg of sodium citrate, 13.3 m9 of aspartic acid, and 147 μl of glutamic acid was used as a standard sample, and 20 μl of it was injected and analyzed. The results are shown in FIG.

However, other analysis conditions are as follows.

(1) Leading electrolyte: 0.01M hydrochloric acid 0.2
β-alanine (pH 3,6
) (2) Terminal electrolyte: 0.01M caproic acid (
3) Electrophoresis current: 100μA, compensation pressure: 5-15KV) (4
) Capillary tube: Inner diameter 0.57m1φ x length 0C
rrL (5) al: 13 mm, a2: 2-3 mm (see Figure 2) (6) Microsyringe needle: outer diameter 0.5
φ(7) Temperature: room temperature, and 30 seconds after aspartic acid detection, the passage door 19 was opened and the electrophoresis current was cut off), and then at time t: 30 seconds, Microsirisin M was transferred to collect 7IL1. The sample was then injected into the analyzer 1 again and analyzed. The results are shown in FIG.

Furthermore, the time t from turning off the electrophoresis current to fractionation was changed to 2 minutes, 10 minutes, 30 minutes, 1 hour, and 3 hours, and the same analysis operation was performed. is shown in Figure 5.

In addition, here, the results for a state in which the electrophoretic current was applied for a time of 20 are also included.

As described above, until time t: about 30 minutes after turning off the electrophoresis current, there is no significant difference in the composition of the aliquoted solution compared to when the electrophoresis current is not turned off. It turns out that the method is also effective.

[Brief explanation of the drawing]

FIG. 1 is a functional explanatory diagram showing an example of a capillary electrophoresis device for carrying out the preparative separation method in electrophoretic analysis according to the present invention, FIG. 2 is an enlarged sectional view of a preparative cell during preparative separation, Figure 3 is a potential gradient graph showing the results of an experiment without fractionation using this device, Figure 4 is a graph corresponding to the previous time when a similar experiment was performed using a fractionated sample, and Figure 5 is a graph of electrophoresis. It is a graph showing the composition of the fractionated liquid when the time t from turning off the electric current until the fractionation is changed. 1... Capillary type isotachophoresis analyzer, 2...
... Terminal liquid electrode tank, 3 ... Sample injection port, 4 ... Preparation cell, 5 ... Capillary tube, 6 ... Leading liquid Electrode tank, 7
...High voltage power supply circuit.

Claims (1)

[Claims] 1. A bent part is provided in the middle of the migration tube in the main body case, and a septum and a detector are provided at this bent part and in front of the bent part, respectively, and on one side of the main body case corresponding to this septum. A door is provided to allow the preparator to pass through, and a detector detects the zone of single component ions migrating while separating them in the electrophoresis tube using electrophoresis.Based on this detection signal, the door of the main body case is opened and the When the door is opened, the switch of the high-voltage power supply circuit that supplies a constant current to the leading and terminal electrolyte tanks attached to both ends of the body movement tube is opened to stop the migration, and the preparator is opened within a predetermined time after stopping. A preparative separation method in electrophoretic analysis characterized by inserting a sample into an electrophoresis tube through an opening in a door and separating a fixed amount of a target component substance.