JPS6221060A - Liquid chromatograph electrophoresis apparatus - Google Patents

Abstract

PURPOSE:To enable simple and high-accuracy separation and analysis of specimen, by allowing effluent liquid obtainable from the liquid chromatography to drip onto a supporting body for the direct electrophoresis in a proper quantity, and the supporting body to slide crosswise to the phoresis direction per each specified time. CONSTITUTION:At the lower reach of a UV detector 5, the dripping exhaust unit of effluent liquid by the liquid chromatography with flow-rate valve 71 and fine tube 7 through a 3-way electromagnetic valve 6. And, its dripping port is positioned on one end of the flat surface part of a cellulose acetate film of the elactrophoresis part. On the other hand, supporting column 13 for displacement and displacement means by a gear set 14 are installed on a supporting stand 12, and as the stand 12 is made to slide along the column 13 by operation of a motor 15, a spot from the dripping exhaust unit is made to displace along a side of the flat surface part of the cellulose acetate film 8 and crosswise to the phoresis direction. Accordingly, an image separated and flowed out by the liquid chromatographic unit can be subjected to the direct electrophoresis separation and a high-accuracy separation and analysis with respect to each component becomes available by sliding of the supporting body.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial Application Field This invention relates to a liquid chromatography-electrophoresis apparatus. More specifically, the present invention relates to a liquid chromatography-electrophoresis device that can continuously perform chromatographic separation and electrophoretic separation and is useful for separating and analyzing various ionic and nonionic components.

(b) Conventional techniques Liquid chromatography and electrophoretic analysis are widely used as separation and separation analysis methods for various samples.

The former separates mixed components based on distribution to the stationary phase, adsorption, ion exchange properties, etc., while the latter separates based on the mobility of target ions in the electrolyte. They are appropriately selected and used depending on the physical and chemical properties of the component to be analyzed.

(c) Problems to be solved by the invention However, when separating and analyzing a sample containing a mixture of ionic and nonionic substances, there are components that cannot be sufficiently separated in principle no matter which separation method is used. In such cases, both methods must be used together. Furthermore, when identifying substances, etc., and separating and analyzing substances with similar physical properties with high precision, it is required to use both of these methods in combination.

Regarding these points, for example, a proposal has been made to perform electrophoresis and chromatographic separation at the same time by filling a chromatography stationary phase in an electrophoresis tube (Japanese Patent Publication No. 58-84).
Publication No. 66). However, this method uses both methods together for one-dimensional separation, and does not fully utilize the individual characteristics of each separation method.

On the other hand, a method of using chromatography in the − dimension and performing zone electrophoresis in the two dimensions is also considered, and in practice, separation and recovery are performed using a-layer chromatography, and electrophoresis is performed in the perpendicular direction. Although some proposals have been made, ′
Since ionization by A-layer chromatography itself is not that good, the final separation accuracy cannot be said to be particularly good, and it is difficult to use anything other than a thin layer of silica gel or canned paper as a support for electrophoresis. There was a point.

The present invention has been made in view of the above situation, and particularly, it is an object of the present invention to provide an analyzer that can easily perform high-precision separation analysis of a sample containing a mixture of ionic and nonionic substances.

(d) Means and operation for solving the problem Thus, according to the present invention, there is provided a liquid chromatograph section in which an eluent supply section, a sample injection section, and a separation column are connected and set in this order, and a liquid chromatograph section for zone electrophoresis. It consists of an electrophoresis section consisting of an abutment on which a flat support is placed and is equipped with an electrophoresis electrode and an electrolyte storage tank, and the separated eluate from the liquid chromatography section is dropped onto the flat support. The present invention is characterized in that a drip discharge section that can be moved is attached downstream of the separation column, and a moving means that can move the support in a direction parallel to one side of the flat support and perpendicular to the electrophoresis direction. A liquid chromatograph-electrophoresis device is provided.

This invention is configured so that an appropriate amount of eluate obtained by liquid chromatography is dropped onto a support for direct electrophoresis onto the electrophoresis starting point, and the support is slid at predetermined intervals at right angles to the direction of electrophoresis. It is designed so that it can be used. As a result, the two fractions that are separated and eluted based on distribution, adsorption, or ion exchange effects in the liquid chromatograph section can be directly subjected to electrophoretic separation, and by sliding the support, the electrophoresis of each fraction can be performed. Electrophoretic separation can be easily performed. Therefore, high-resolution separation analysis of each contained component becomes possible.

The dripping discharge section into which the separated eluate from the liquid chromatography section can be dripped can be constructed by providing a suitable flow rate control means on the drain of a conventional liquid chromatography device and setting the discharge port downward. Examples of such flow rate control means include a flow rate regulating valve and a branch pipe. The dripping flow rate in the liquid chromatograph section is usually 0.5 to 21! /minute is appropriate. However, at this flow rate, the number of spots on the flat support of the electrophoresis section is too large, which may cause problems in electrophoretic separation. In such a case, if the dropping flow rate is set to about 0.11/min, the separation time by the liquid chromatograph section will become longer, so the flow ω is kept at the normal flow rate and the flow rate is increased to 115 to 1/20. The branched dropping liquid is spotted onto an electrophoresis support.

Also, if a capillary liquid chromatograph section is used,
The dropping liquid can be spotted directly onto the support.

The moving means of the support of the electrophoresis analysis section is configured to move at least the flat support parallel to one side thereof and in a direction perpendicular to the electrophoresis direction. Thereby, the position of the spot dropped onto the support from the dropping discharge part can be moved at predetermined time intervals without changing the migration distance. Generally, it is preferable to position the drip outlet and the support so that the drop spot can move along one side of the support. However, the central part of the support may be positioned so that it can move at right angles to the electrophoresis direction, and in this case, the anionic component and cationic component may be caused to migrate in the direction of the positive and negative electrodes, respectively, from that spot. One favorable point 11 that can be done! ! It's like that. From this point of view, a second movement means that can set the initial position of the spot according to the purpose, that is, a means that can move the spot position parallel to the electrophoresis direction, is attached, and the movement path is set appropriately on the side or the center. It can also be configured to do so.

On the other hand, as an eluent for liquid chromatography,
It is suitable to use a phosphate buffer and acetonitrile because it does not adversely affect the affinity with the electrolytic solution for electrophoresis faJ or the electrophoretic properties. Therefore, liquid chromatography is usually performed in reversed phase mode, and the separation columns that can be used include so-called ODS columns, 08 columns, TMS columns, and ion exchange separation columns WAX.

In addition, as the flat support in the electrophoresis section, rectangular polyacrylamide gel membranes, cellulose acetate membranes, filter paper, etc. used in ordinary zone electrophoresis analysis are used, and the size depends on the analysis items and the number of electrophoresis spots. Determined accordingly. Further, as the support base itself on which the flat support is placed, a known support base that is equipped with an electrolyte storage tank and an electrophoresis electrode can be used.

Note that it is preferable to attach a detector between the separation column and the dripping/discharging section, and various kinds of detectors commonly used in high-performance liquid chromatographs can be used. Furthermore, a switching valve can be provided before the dripping discharge 815 to temporarily stop dripping onto the support. Such a switching valve removes unnecessary eluent (mobile phase only, etc.),
Both parts or only a part thereof can be selectively dropped onto the support. In particular, a control unit is provided which detects the presence or absence of a peak with the detector and accordingly drives a switching valve to drop only both portions onto the support and intermittently drives the moving means in accordance with the switching of the peak. It is also a preferable mode to automatically control.

(E) Embodiment (1) shown in FIG. 1 is an explanatory diagram of the configuration of an embodiment of the liquid chromatograph-electrophoresis apparatus of the present invention. In the figure, the liquid chromatograph-electrophoresis device (1) is
The eluent tank (21: phosphate buffer with pH 6, 8), liquid pump (2, sample injection part (3), separation column (4; ion exchange separation column WAX) and U detector (5) are connected to this A cellulose acetate membrane (8) for zone electrophoresis is placed on the support surface, which is equipped with a high-performance liquid chromatograph section which is connected to pipes in order, an electrode (9) and an electrolytic solution tank (11: hydrochloric acid-Tris solution). It basically consists of an electrophoresis section having a planar electrophoresis surface.

A three-way electromagnetic valve (6) is located downstream of the U■ detector (5).
), the eluate dripping outlet is composed of a flow valve (11) and a tip orifice-like thin tube (7), and the dripping port is connected to the flat part of the cellulose acetate membrane (8) in the electrophoresis section. The position is set corresponding to one end of. On the other hand, the support stand (+21) in the electrophoresis section consists of a movement support (131) and a gear (141), and a motor (1
A moving means is attached to the drive of the support 51 to slide the support color along the direction of the electrophoresis. The spot from the drip discharge section is configured to be movable.

On the other hand, the port is a control unit that opens and closes the three-way electromagnetic valve (6) based on the signal from the detector (5) and the motor 05]
ノON-OF F11111Tru.

In the apparatus (1), both components separated in the liquid chromatograph section are detected as peaks by the detector (5). The detection signal from the first peak is determined by the clever 1 part (16
1 first switches the three-way solenoid valve (6) to the solid line side.

At this time, the drip opening of the drip discharge part is set at one corner of the support, and the first two portions are dripped to give spot A. A predetermined voltage is applied between the electrodes (9)a○) in the electrophoresis section, and the spot A is then subjected to electrophoresis and separated by the mobility of the ionic substance, resulting in a ferogram of the anion component. (81) is obtained. In addition,
If separation of cationic components is intended, the polarity of the electrode (9) color may be reversed. Further, when it is intended to separate both anion and cation components, initial settings may be made so that a spot is formed in the center between both electrodes as described above.

On the other hand, the first peak has ended and only the mobile phase is detected by the detector (5).
When detected, the control unit (16) switches the three-way electronic valve (6) to the dashed line side based on the signal level (background level) to guide the detected eluate to the drain (61). Next, when the second peak is detected, the control unit (16) again moves the cellulose acetate membrane (8) in the direction of the arrow by a predetermined distance (for example, around 0.5 part 1) by turning the valve (6) ONL. As a result, the second two portions are dropped, forming spot B next to spot A, and subsequently subjected to electrophoresis in the same manner as above. Thereafter, the electromagnetic valve (6) is switched and the motor (151 is driven/stopped) in the same manner depending on the presence or absence of a peak, and spot C... is placed on the side of the cellulose acetate film (8). and are each successively subjected to electrophoretic separation.

Note that the nonionic components remain retained on each spot, making it possible to easily distinguish between ionicity and nonionicity of each chromatographically separated component.

(F) Effects of the Invention As described above, the apparatus of the present invention is capable of performing liquid chromatography and electrophoretic separation substantially simultaneously, and is particularly useful for samples containing a mixture of ionic and nonionic substances. This enables high-precision separation analysis easily. Since the characteristics of liquid chromatography and electrophoretic separation are utilized and linked through a specific configuration, the ionization performance of each is basically not inhibited in any way, and the flat support in the electrophoresis section is also Various known materials can be selected and used.

Therefore, it is extremely useful for separating and analyzing samples containing a large number of ionic and nonionic substances, such as proteins, nucleic acid-related substances, organic acids, and amines.

[Brief explanation of the drawing]

FIG. 1 is a configuration explanatory diagram showing one embodiment of a liquid chromatograph-electrophoresis apparatus of the present invention. (1)...Liquid chromatograph-electrophoresis device, (a...liquid pump, (3)...
...sample injection section, (4) ...separation column,
(Sword...tubule, (71)...flow valve, (8)...cellulose acetate membrane, (9)
001...electrode, 0t...electrolyte storage tank,
021...Abutment, (13)...
Pillar, ⑋)...Gear, (151...
···motor.

Claims (1)

[Claims] 1. A liquid chromatograph section consisting of an eluent supply section, a sample injection section, and a separation column connected in this order, a flat support for zone electrophoresis, and an electrode for electrophoresis and an electrolyte storage tank. An electrophoresis section consisting of an abutment provided with a column, and a dripping discharge section capable of dripping the separation eluate from the liquid chromatography section onto the flat support is attached downstream of the separation column, and the above-mentioned flat support A liquid chromatograph-electrophoresis apparatus characterized in that a moving means capable of moving the support in parallel to one side of the support and perpendicular to the migration direction is provided.