CN1372138A - Electrophoresis focusing concentrator - Google Patents

Abstract

It relates to a device for analyzing materials by electrophoresis. Two electrolyzers are provided, and the electrolyzers are connected by pipes, and the electrolyzers are separated from the pipes by solid ion conductors. It is designed using the principle of aggregation electrophoresis concentration, with the purpose of focus concentration preparation. Focusing electrophoresis has a fast concentration speed, a simple device, and a good concentration effect (up to two orders of magnitude). These all illustrate that the present invention has great development prospects, and can be used in analytical chemistry (for example, capillary electrophoresis as a pre-concentration device) to reduce the detection limit and make the detection of trace substances possible; it can also be used to purify some valuable biochemical substances and precious rare substances.

Description

An electrophoretic focusing and concentrating device

The invention relates to a device for analyzing materials by electrophoresis.

In the mid-19th century, Wiedeman and Buff reported the phenomenon of charged particles moving in solution under the action of an electric field. This phenomenon was later called electrophoresis. Electrophoresis not only refers to this phenomenon but also a separation method and technique. Electrophoresis is the technique of separating and identifying charged particles in a mixture. Before the 1920s, the application of electrophoresis technology was limited to zone electrophoresis. It was not until 1923 that isotachophoresis appeared, and rare earth metals and some simple acids were successfully separated. With the continuous development of electrophoresis technology, there are mainly four kinds of separation principles, namely, zone electrophoresis, mobile interface electrophoresis, isotachophoresis and isoelectric focusing electrophoresis. Electrophoresis, as a separation technique, is being used more and more widely. In particular, high-performance capillary electrophoresis (HPCE), which can apply extremely high voltages, achieves unprecedented high resolution and separation efficiency with extremely short separation times. It has been widely researched and applied in today's chemistry, molecular biology and biochemistry. However, there are three technical difficulties in capillary zone electrophoresis: (1) sample injection is difficult; (2) on-site pre-concentration cannot be realized; (3) the detection method is limited due to the characteristic of separation zone migration, which hinders the detection of array capillary tube electrophoresis. The development of electrophoresis. Patent application CN1193107A discloses a countercurrent focusing electrophoresis device, which is provided with an electrophoretic channel filled with electrophoretic solution, a solid-phase ionic conductor parallel to the channel and in contact with each other, an electrophoretic solution driver, positive and negative electrode chambers and positive and negative electrodes, DC power supply. The two ends of the channel are respectively connected to the electrophoretic solution driver and the electrode chamber, and the ratio V _X /ε _X of the linear velocity V _X of the electrophoretic solution to the electric field strength ε _X of the electrophoretic solution driving ions to swim in the X direction is a monotone function of X.

The present invention aims to provide an electrophoresis focusing and concentrating device capable of concentrating charged ions with high efficiency. It is mainly used for pre-concentration sample injection in the early stage of separation and analysis of trace substances, and can be independently used for the purification of some valuable biochemical reagents. Improving current problems in capillary electrophoresis.

The invention is provided with two electrolyzers, the electrolyzers are connected by tubular objects, and the electrolytic cells are separated from the tubular objects by solid ion conductors. When concentrating anions, use a solid cation conductor to separate the anode tank from the tube; when concentrating cations, use a solid anion conductor to separate the cathode tank from the tube.

In application, take the concentration of anions as an example. When the device is powered on, the cations can pass through the solid cation conductor to reach the cathode tank smoothly under the drive of the electric field, while the anions will move to the positive tank under the action of the electric field, but because they cannot penetrate It is a solid cation conductor, so it can only gather in small holes to achieve the effect of concentration.

Existing electrophoresis techniques can be divided into four main types: zone electrophoresis, moving interface electrophoresis, isotachophoresis, and isoelectric focusing. The separation of various ions is mainly due to the different mobility of the ions, while isoelectric focusing relies on their different isoelectric points. In terms of application, it can be divided into two categories: detection type and preparation type. The former is used for separation detection and identification, while the latter is used for the purpose of separation and preparation of certain components.

A zonal electrophoresis system is characterized in that the entire system (positive electrode, negative electrode, separation chamber) is filled with the same electrolyte. Generally, the concentration of the background electrolyte is higher than the sample concentration, so it provides a constant pH and potential gradient. When energized, sample ions and electrolyte ions move at specific speeds. If the mobilities of different ions in a sample are very different, then over time the different ions will separate from each other. The disadvantage of this detection method is that the detection cannot be performed until after the separation step is completed. In addition, it takes a long time for processing to go through several additional steps, often causing troublesome problems such as zone diffusion.

Mobile interface electrophoresis uses a small hole tube with positive and negative electrodes connected at both ends as a separation chamber for anion separation. The positive electrode tank and the fine-pore tube are filled with the same electrolyte, and the anion of the selected electrolyte has a higher mobility than all the anions to be separated, and the sample is added to the negative electrode tank. The anions move to the positive electrode, but the sample anions cannot go beyond the pre-conductivity electrolyte, and the flow of various anions in the sample is also uneven.

The characteristic of isotachophoresis is that the anions of the leading electrolyte filled with the fine-pore tube and the positive electrode tank must occupy a mobility greater than that of all the anions to be separated, and the negative electrode tank is filled with the trailing electrolyte, and its anion mobility is smaller than that of all anions. The sample is added between the leading electrolyte and the trailing electrolyte. After electrifying for a period of time, various anions in the sample are arranged in the order of their effective mobility. At the same time, the sample zone is between the leading electrolyte and the trailing electrolyte. The first sample zone is the anion with the largest effective mobility in the sample, and the last sample zone is the anion with the smallest effective mobility in the sample. So far, The sample will not be further separated. The system enters a steady state, in which case we can consider it as a constant velocity separation system. At this stage all zones are connected and have the same rate of migration.

Ampholytes have zero net charge at the isoelectric point and will not migrate under the action of an electric field. A pH gradient buffer is placed in the separation chamber, and the pH value is transferred from one end of the separation chamber to the other end. The amphoteric mixture is injected into this system, and an electric field is applied to this system, and each substance moves to a fixed position, and the pH value of this position is equal to the isoelectric point value pI of the substance, that is, the substance is in this place focus. Different substances have different pI values, so their focus points are also different, and then the purpose of separation is achieved. However, the scope of application of this method is small, and it is currently limited to the separation of proteins and peptides with amphoteric characteristics.

The various electrophoresis techniques mentioned above have many disadvantages. For example, the sample loading zone of zone electrophoresis is required to be very narrow, sample concentration and sample separation cannot be carried out at the same time, and the separation zone is always in dynamic state. and higher response speed requirements. Isoelectric focusing is only suitable for the separation of ampholyte samples, and the buffer solution can only be a mixture of ampholyte in a specific range, and the price is relatively high. Isotachophoresis requires the selection of a discontinuous buffer system. After the separation is stable, the samples form sharp zones and are closely arranged. For detection, spacers may need to be added. The separation effect of mobile interface electrophoresis is not good, and the sample cannot be separated independently.

The present invention is designed by utilizing the principle of aggregation electrophoresis concentration, and aims at focusing concentration preparation. Focusing electrophoresis has a fast concentration speed, a simple device, and a good concentration effect (up to two orders of magnitude). These all illustrate that the present invention has great development prospect, can be used in analytical chemistry (for example capillary electrophoresis is made pre-concentration device) and reduces detection limit, makes the detection of trace substance possible; Also can be used for purifying some valuable biochemical substances and precious rare substances.

Fig. 1 is a structural schematic diagram of the present invention.

Fig. 2 is a schematic diagram of the device for testing the concentration effect by using the optical fiber detection method.

Fig. 3 is a schematic diagram of the device for testing the concentration effect by microelectrode cyclic voltammetry.

Fig. 4 is a schematic diagram of a device for testing concentration effect by colorimetry.

Figure 5 is a graph showing the concentration effect tested by the colorimetric method.

The following examples will further illustrate the present invention by taking concentrated anions as an example in conjunction with the accompanying drawings.

As shown in Figure 1, the present invention is provided with 2 electrolyzers: anode tank 1 and cathode tank 2, is connected with channel pipe 3 between electrolyzer 1 and 2, uses a solid cation conductor between channel tube 3 and anode tank 1 4 Separate the electrolyzer. After energization, the cations can pass through the solid cation conductor 4 and reach the cathode tank 2 smoothly under the drive of the electric field, while the anions will move to the anode tank 1 under the action of the electric field, but because they cannot pass through the solid cation conductor 4, they can only Gather at the joint 5 between the channel tube and the solid-state ion conductor, thereby achieving the effect of concentration. A concentrated liquid outlet pipe 6 is provided at the connection 5, and when the concentration reaches the requirement, the concentrated liquid can be slowly drawn out from the concentrated liquid outlet pipe 6.

During electrophoresis, the electrolysis of the positive and negative electrodes will produce H ⁺ and OH ^- , which will change the pH of the electrophoresis solution and cause some chemical changes (or change the charge state, or change the color) of some ions to be concentrated. Ions have different pH requirements, and the selected buffer solutions are also different. In this example, bromophenol blue solution and sodium sulfate solution can be used as anion and cation solutions. In order to prevent the influence of other ions, we use C ₆ H ₁₃ NO ₂ buffer solution at the cathode, and 1:3.5 KH ₂ PO ₄ and _{Na2HPO4 buffer} solution. The pH on both sides is controlled around 9.0.

When the concentrate is cation, a solid anion conductor is used, and it is arranged between the cathode tank 2 and the channel pipe 3 .

Fig. 2 provides the schematic diagram of the device adopting the optical fiber detection method to test the concentration effect, the anode tank 1 and the cathode tank 2 are separated by the solid ion conductor 4, the optical path of the light source 7 and the detector 8 is introduced and drawn out by the optical fiber 9, and the concentrated area is used as the optical fiber. detection pool.

Figure 3 shows the schematic diagram of the device for testing the concentration effect by microelectrode cyclic voltammetry. The experimental object was changed to redox species such as K ₃ Fe(CN) ₆ , and the concentration of Fe(CN) ₆ ^3- in the concentrated area was detected by a micro-platinum electrode. A channel tube 3 is arranged between the anode tank 1 and the cathode tank 2 , and the solid ion conductor 4 separates the anode tank 1 from the channel tube 3 . The device is additionally provided with a ring-shaped auxiliary electrode 10 , a reference electrode 11 , and a micro-platinum electrode 12 .

Figure 4 shows the schematic diagram of the device for testing the concentration effect by colorimetry. A small opening 13 is opened at the concentrated place, and a glass tube is used to export the concentrated solution, and then a series of bromophenol blue solutions with different concentrations are placed in the outlet for colorimetry. The instrument and reagents use a high-voltage constant-current power supply 14 with a constant current of 0-1mA and an output voltage of 0-1000V. Anode solution: 10 ^-3 MNa ₂ SO ₄ solution; 1:3.5KH ₂ PO ₄ and Na ₂ HPO ₄ buffer solution (pH≈9.0). Cathode solution: series bromophenol blue solution; C ₆ H ₁₃ NO ₂ buffer solution (pH≈9.0). During the experiment, the outlet tube of the concentrated solution can continuously draw out the concentrated solution with a micro suction pump. The outlet nozzle is connected to a glass tube (inner diameter ≤ 0.5mm), and the nozzle at the other end is blocked. The concentrated solution sinks into the (convection, diffusion) tube due to gravity, and the amount of deposition increases with time. Colored samples are used, which are easy to observe, and can also be compared with sample glass tubes (the diameter of the tube is the same as that of the outlet tube). During the experiment, a constant current of 300 μA was given, and the experimental concentration phenomenon at different times was photographed and recorded.

The experimental results show that with the increase of time, the concentration of the outlet liquid is getting higher and higher, and the concentration zone in the tube is getting longer and longer. Finally the whole tube can be filled. It can also be seen from Figure 5 that the device can well concentrate some liquids to be tested (up to two orders of magnitude). In Figure 5, bromophenol blue solution is selected, a is 10 ^-5 M (initial concentration), b is 10 ^-4 M, c is 10 ^-3 M, and d is the concentration of bromophenol blue in the outlet tube.

Claims (3)

1. An electrophoresis focusing and concentrating device is characterized in that it is provided with 2 electrolyzers, and the electrolyzers are connected with tubular objects, and the electrolyzers are separated from the tubular objects with solid ion conductors. 2. An electrophoretic focusing and concentrating device as claimed in claim 1, characterized in that when concentrating anions, a solid cation conductor is used to separate the anode tank from the tube. 3. An electrophoretic focusing and concentrating device as claimed in claim 1, characterized in that when concentrating cations, a solid anion conductor is used to separate the cathode tank from the tube.

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