WO2015139156A1 - 全自动高精度毛细管电泳仪 - Google Patents

全自动高精度毛细管电泳仪 Download PDF

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Publication number
WO2015139156A1
WO2015139156A1 PCT/CN2014/000473 CN2014000473W WO2015139156A1 WO 2015139156 A1 WO2015139156 A1 WO 2015139156A1 CN 2014000473 W CN2014000473 W CN 2014000473W WO 2015139156 A1 WO2015139156 A1 WO 2015139156A1
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Prior art keywords
capillary
port
electrophoresis
bottle
flow path
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PCT/CN2014/000473
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English (en)
French (fr)
Inventor
阎超
姚冬
张琳
李静
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阎超
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Priority to JP2016552919A priority Critical patent/JP6208378B2/ja
Priority to US15/128,032 priority patent/US9903835B2/en
Publication of WO2015139156A1 publication Critical patent/WO2015139156A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44743—Introducing samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44708—Cooling
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus

Definitions

  • the invention relates to capillary electrophoresis of substances, in particular to a fully automatic high-precision capillary electrophoresis apparatus, belonging to the technical field of analysis and testing.
  • Capillary electrophoresis began in the 1980s. During the analysis, the capillary is filled with a buffer solution, and high voltage is applied across the capillary (generally 0 to +30 kV or to -30 kV). The electroosmotic flow in the capillary causes the solution to flow toward the detector, which also drives the capillary. All substances flow to one end of the detector. The substances in the solution have different electrophoretic enthalpy, that is, the ratio of the charged charge to the molecular weight (charge-to-mass ratio) is different, so the speed of reaching the capillary end detector is different, thereby obtaining separation and detection.
  • the capillary electrophoresis system uses the principle that the charged material in the capillary is moved under the action of the electric field to achieve the purpose of separation.
  • the main components include a long tubular capillary, a liquid container at both ends of the capillary, an electrode, a high voltage power supply, Detector and a data output and processing unit.
  • capillary injection There are two general methods of capillary injection: fluid mechanics and electromigration injection. Hydrodynamic injection can be achieved by siphoning, pressurizing at the injection end, or detecting H3 ⁇ 4 evacuation. Electromigration injection is the application of a voltage. By electric field, the sample is injected by electromigration and/or electroosmotic flow of the sample ions. Capillary.
  • the above two injection methods have the following disadvantages: 1. Since both methods use the "draw" method to complete the injection, both the capillary is poured into the sample vial, pressure or voltage is applied, and then returned to the solution bottle. Separation is carried out, and the amount of sample entering the capillary can only be estimated by rough estimation. Therefore, the accuracy of the two injection methods is lacking. 2.
  • the capillary electrophoresis instrument has a very small injection volume, which is in the nanoliter (nl) class.
  • the conventional injection method used in the existing capillary electrophoresis apparatus cannot accurately quantify such a small volume, and the capillary end draws the injection method.
  • the injection repeatability error is large and it is difficult to use for quantitative measurement.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art, and provide a fully automatic high-precision capillary electrophoresis apparatus to realize automatic quantitative injection, improve the accuracy, reproducibility and reliability of injection; Seek the phenomenon of mutual contamination between the sample; increase the injection speed, shorten the injection time, minimize the outside air into the injection flow path; at the same time realize the automatic cleaning and balance of the capillary separation column with different reagents.
  • a fully automatic high-precision capillary electrophoresis apparatus includes an electrophoresis system including an electrophoresis separation capillary outlet inserted into a buffer liquid discharge bottle, a column oven and a detector sequentially connected to the electrophoresis separation capillary, and a high voltage power supply One pole is inserted into the buffer liquid discharge bottle, and the other pole is connected to the electrophoresis separation capillary through an electrical isolation device and a high voltage electric field is formed in the electrophoresis separation capillary, which is characterized by:
  • the capillary electrophoresis apparatus further includes an injection flow path connected to the electrophoresis system and an automatic sampling flow path connected to the injection flow path;
  • the injection flow path includes a split waste liquid bottle, a four-way micro injection valve and a buffer injection pump respectively connected to three interfaces of a four-way connector;
  • the four-way micro-injection valve can accurately quantify, and is used for quantitative injection into the electrophoresis separation capillary, including four fixed pipeline ports of S port, W port, P port and C port, and a rotatable built-in quantitative a ring, the C port is connected to the electrophoresis separation capillary, the P port is connected to the four-way connector, the built-in loop is provided with a bypass passage and a quantitative passage having a fixed volume, along with the built-in loop Rotating, the bypass path and the quantitative path are switched between connecting the S port and the W port and connecting the P port and the C port;
  • the automatic sampling flow path includes a sampling needle, a cleaning liquid bottle, a reagent bottle, and a six-terminal liquid dispenser and a syringe pump connected by two ends of the buffer tube having a quantitative function;
  • the syringe pump is equipped with a three-terminal distribution valve capable of switching the exhaust and cleaning functions, and the three valve interfaces of the three-terminal distribution valve are respectively connected to the cleaning liquid bottle, the autosampler waste liquid bottle and the buffer tube;
  • the six-terminal liquid dispenser has a fixed interface and six distribution interfaces that can be alternately connected to the fixed interface, the fixed interface is connected to the buffer tube, and the six distribution interfaces are respectively connected to the sampling needle and the four-way connection An interface of the device, a four-port micro-injection S port, and three reagent bottles equipped with different reagents for cleaning and balancing the electrophoresis separation capillary;
  • a cleaning tank for cleaning the sampling needle is connected between the cleaning liquid bottle and the autosampler waste liquid bottle, and the sampling needle is capable of switching into the cleaning tank or the sample tray containing different samples;
  • the autosampler waste bottle is connected to the W port of the four-way micro injection valve.
  • the electrophoresis system further includes a balance waste liquid bottle, and the outlet of the electrophoresis separation capillary can be switched to connect the balance waste liquid bottle or the buffer liquid discharge bottle.
  • the volume of the quantitative passage of the four-way micro-injection valve is from InL to 20 nL Any volume between.
  • a capillary pressure sensor for detecting the working pressure of the pipeline is connected between the buffer injection pump and the four-way connector, and the connection between the three-end distribution valve and the buffer tube of the syringe pump is detected.
  • the sample tray has a constant temperature and refrigeration structure.
  • the cleaning liquid bottle is provided with a cleaning liquid which is ethanol or deionized water.
  • the functions of all of the components of the capillary electrophoresis apparatus are automatically controlled by a computer program.
  • connection between the four-way connector and the split waste bottle is used to balance the internal pressure of the electrophoretic separation capillary.
  • the detector is a UV detector or other capillary on-column detector.
  • the automatic sampling flow path of the present invention is used in combination with a syringe pump with a three-terminal distribution valve and a six-terminal liquid dispenser, and the sample is quickly drawn through the large-diameter pipe.
  • the passage of the six-end liquid distributor is switched, and the sample is driven into the four-way micro-injection by positive pressure, thereby overcoming the shortcomings such as slow injection speed caused by negative pressure injection, and greatly shortening the injection.
  • the present invention employs a nano-scaled four-way micro-injection valve with a built-in loop, the quantitative volume of which is any determined volume between InL and 20 nL, through which the precise quantitative injection is completed, the present invention
  • the injection method overcomes the shortcomings of the traditional capillary end extraction or gas pressure injection mode, which can not complete such a small volume accurate injection, realizes automatic quantitative injection, improves the reproducibility of the injection, and the injection repeatability is relatively The standard deviation is less than 3%.
  • the fully automatic high-precision capillary electrophoresis apparatus of the invention has the advantages of fast injection speed, good quantitative accuracy, high precision, good reproducibility and easy industrialization, and realizes automation of sample injection, capillary cleaning and balance. Can be widely used for capillary electrophoresis analysis of different substances.
  • Figure 1 is a schematic view of the structure of the present invention.
  • FIG. 1 is one of the schematic diagrams of the working process of the present invention.
  • Figure 3 is a second schematic diagram of the working process of the present invention.
  • Figure 4 is a third schematic diagram of the working process of the present invention.
  • Figure 5 is a fourth schematic diagram of the working process of the present invention.
  • Figure 6 is a fifth schematic diagram of the working process of the present invention.
  • Figure 7 is a sixth schematic diagram of the working process of the present invention.
  • Figure 8 is a seventh schematic diagram of the working process of the present invention.
  • Buffer syringe pump 1 Buffer syringe pump, 2 capillary pressure sensor, 3 four-way connector, 4 shunt waste bottle, 5 four-way micro injection valve, 51 quantitative channel, 52 bypass path, 6 electrical isolation device, 7 electrophoresis separation capillary, 8 column thermostat, 9 high voltage power supply, 10 detectors,
  • the fully automatic high-precision capillary electrophoresis apparatus of the invention is used for capillary electrophoresis analysis of different substances.
  • the fully automatic high-precision capillary electrophoresis apparatus includes an electrophoresis system, an injection flow path, and an automatic sampling flow path.
  • the injection flow path is connected to the electrophoresis system, and the automatic sampling flow path and the inlet are The sample flow path is connected.
  • the electrophoresis system includes an electrophoresis separation capillary 7, a column oven 8, a detector 10, a high voltage power source 9, an electrical isolation device 6, a buffer liquid discharge bottle 11, and a balance waste liquid bottle 12.
  • the electrophoresis separation capillary 7 is a place where the substance is separated by capillary electrophoresis, and is also a core original component of the separation analysis of the system, and the outlet is inserted into the buffer liquid discharge bottle 11;
  • the column temperature tank 8 controls the temperature of the electrophoresis separation capillary 7 , mainly used to emit Joule heat generated during some electrophoretic separation, and to prevent low temperature crystallization of the internal material of the capillary during the capillary electrophoresis separation experiment;
  • the detector 10 is used for detecting the separated signal, which may be ultraviolet a detector or other capillary column detector, the column oven 8 and the detector 10 are sequentially connected to the electrophoresis separation capillary 7;
  • the buffer liquid discharge bottle 11 is an outlet of
  • the injection flow path includes a buffer injection pump 1, a capillary pressure sensor 2, a four-way micro injection valve 5, a four-way connector 3, and a shunt waste liquid bottle 4.
  • the buffer injection pump 1 is configured to supply a supplemental buffer and a certain pressure to the electrophoresis separation capillary 7;
  • the capillary pressure sensor 2 is configured to detect a working pressure of the electrophoresis separation capillary and the bypass line;
  • the split waste liquid bottle 4 is for accommodating the buffer which is branched by the four-way connector 3.
  • the four-way micro-injection valve 5 has an accurate quantitative function, including four fixed pipeline ports of S port, W port, P port and C port, and a rotatable built-in loop, the built-in loop is provided with a side a path 52 and a quantity of passages 51 having any fixed amount of volume between InL and 20 nL, such as 4 nL, 10 nL or 20 nL, for quantitative sampling of nano-upgrades;
  • the rotation of the loop, the bypass passage 52 and the metering passage 51 are switched between communicating the S port and the W port and the communication P port and the C port, specifically, when in the liquid filling position, the quantification
  • the passage 51 communicates with the S port and the W port
  • the bypass passage 52 communicates with the P port and the C port.
  • bypass passage 52 communicates with the An S port and a W port, and the quantitative passage 51 communicates with the P port and the C port; a C port of the four-way micro injection valve 5 is connected to the electrophoresis separation capillary 7 for separating the electrophoresis capillary 7 into Quantitative sampling.
  • the three interfaces of the four-way connector 3 are respectively connected to the buffer ports of the buffer injection pump 1, the split waste liquid bottle 4 and the four-way micro injection valve 5; the capillary pressure sensor 2 is connected to the buffer Between the syringe pump 1 and the four-way connector 3, a filter is connected between the four-way connector 3 and the P port of the four-way micro-injection valve 5 for filtering large particles of impurities in the sample neutralizing reagent.
  • the four-way micro-injection valve 5 and the electrophoresis separation capillary 7 are protected, and a shunt tube 23 is connected between the four-way connector 3 and the shunt waste liquid bottle 4, and the shunt tube 23 balances the electrophoresis by changing the caliber and length.
  • the automatic sampling flow path includes a six-terminal liquid dispenser 13, a sampling needle 15, a sample tray 16, a washing tank 17, a buffer tube 14, a reagent bottle 20, a sampling flow path pressure sensor 18, a syringe pump 19, a cleaning liquid bottle 21, and Autosampler waste bottle 22.
  • the six-terminal liquid distributor 13 has a fixed interface and six distribution interfaces that can be alternately connected to the fixed interface; the fixed interface is connected to the buffer tube 14, and the six distribution interfaces are respectively connected to the sampling needle 15, One port of the four-way connector 3, the S port of the four-way micro-injection valve 5, and three reagent bottles 20 containing different reagents for washing and balancing the electrophoretic separation capillary 7.
  • the syringe pump 19 is a source of the entire system flow path, and is equipped with a three-terminal distribution valve capable of switching the system exhaust and cleaning functions, and the three valve ports of the three-end distribution valve are respectively connected to the cleaning liquid bottle 21 Self The injector waste bottle 22 and the buffer tube 14 are moved.
  • the sampling flow path pressure sensor 18 is for detecting the working pressure of the sampling flow path, which is connected between the three-end distribution valve of the syringe pump 19 and the buffer tube 14.
  • the cleaning liquid bottle 21 is provided with a cleaning liquid, which is also a mobile phase of the injection flow path, and is generally selected from ethanol or deionized water.
  • the autosampler waste bottle 22 is for containing waste liquid after system cleaning, which is connected to the W port of the four-way micro injection valve 5.
  • the cleaning tank 17 is for simultaneously cleaning the outer wall and the inner wall of the sampling needle 15, and is connected between the cleaning liquid bottle 21 and the autosampler waste bottle 22.
  • the buffer tube 14 has a certain quantitative function for temporarily storing a space including a sample, a injection mobile phase, a reagent, a cleaning liquid and the like in the system; the six ends of the buffer tube 14 and the six-end liquid distributor 13 and The syringe pumps 19 are connected, and by the cooperative operation of the combined members, functions such as system exhaust, cleaning (equilibrium), and automatic quantitative injection can be performed.
  • the sample tray 16 is provided with different samples, which have a constant temperature and refrigeration structure, can keep the sample at a very low temperature, maintain the activity of the biological enzyme, and reduce the volatilization of the sample; the sampling needle 15 has a puncture function and can be worn.
  • the rubber stopper of the vial is drilled deep into the inside of the vial, and the sampling needle 15 can be switched into the cleaning tank 17 or the sample tray 16.
  • the working principle of the fully automatic high-precision capillary electrophoresis apparatus of the present invention is as follows:
  • the outlet end of the electrophoresis separation capillary 7 is switched to the buffer liquid discharge bottle 11, and the four-way micro-injection valve 5 is located at the liquid filling position, that is, the four-way micro-injection
  • the metering passage 51 in the valve 5 communicates with the S port and the W port; the six-terminal liquid distributor 13 is switched to communicate with the sampling needle 15, which is inserted into the sample tray 16, which sucks the "displacement volume"
  • the sample (generally not less than 1.5 times the volume of the sampling needle 15) is filled with the sampling needle 15, and then a certain amount of air is continuously drawn to completely enter the buffer tube 14; thereby rinsing the sampling needle 15 with the sample
  • the inner wall replaces the cleaning fluid and the interference components in the pipeline so that they are not injected into the separation flow path.
  • the six-terminal liquid distributor 13 is switched to connect the S end of the four-way micro-injection width 5, and the syringe pump 19 pushes out the "displacement volume" sample into the built-in quantitative value of the four-way micro-injection valve 5.
  • the sample is filled to the quantitative passage 51 to reach a certain amount, and the sample is flushed to the inner wall of the four-way micro-injection valve 5, and the cleaning liquid and the interference component in the pipeline are pushed out of the W-end of the four-way micro-injection valve 5.
  • the autosampler waste bottle 22 is not injected into the separation flow path.
  • the built-in quantitative ring rotates by 180°, and the four-way micro-injection valve 5 is switched to the position of the liquid injection, that is, the quantitative path 51 filled with the sample in the four-way micro-injection valve 5 communicates with the P Port and C port, Connected to the electrophoresis separation capillary 7; the pressure of the buffer injection pump 1 causes the sample in the quantitative passage 51 to enter the electrophoresis separation capillary 7, and the quantitative electrophoresis analysis of the sample is started; meanwhile, the sampling needle 15 is moved to the cleaning tank 17 in.
  • the built-in loop is rotated by 180°, and the four-way micro-injecting valve 5 is switched back to the filling position, that is, the quantitative path 51.
  • the six-terminal liquid distributor 13 is kept in communication with the S end of the four-way micro-injection valve 5, and the syringe pump 19 cleans the cleaning liquid bottle 21 The liquid is injected into the four-way micro-injection valve 5 to perform a small flow cleaning of the built-in loop.
  • the six-terminal liquid dispenser 13 is switched into a sampling needle 15 inserted into the cleaning tank 17, and the syringe pump 19 injects the cleaning liquid into the sampling needle 15 The sampling needle 15 and the sampling flow path are cleaned.
  • the sixth step referring to FIG. 7, after completion of the capillary electrophoresis analysis, enters the reagent cleaning and equilibrium electrophoresis separation capillary 7 stage; the six-terminal liquid dispenser 13 converts the reagent bottle 20 required for communication, and cleans and balances with the syringe pump 19. The reagent for electrophoretic separation of the capillary 7 is sucked into the buffer tube 14.
  • a seventh step referring to FIG. 8, the outlet end of the electrophoresis separation capillary 7 is switched to the balance waste liquid bottle 12, and the six-terminal liquid distributor 13 is switched to communicate with the four-way connector 3, and the syringe pump 19 is used to be specific.
  • the reagent in the buffer tube 14 is pushed into the electrophoresis separation capillary 7 through the bypass passage 52 of the four-way micro-injection valve 5, and the electrophoresis separation capillary 7 is cleaned and balanced, and the washed waste liquid enters the balance waste. Liquid bottle 12.
  • the six-terminal liquid distributor 13 is kept in communication with the four-way connector 3, and the cleaning liquid in the cleaning liquid bottle 21 is injected into the buffer tube 14 by the syringe pump 19, and then injected into the electrophoresis.
  • the capillary 7 is thereby cleaned of the entire buffer tube 14 and the electrophoresis separation capillary 7.
  • the fully automatic high-precision capillary electrophoresis apparatus restores the initial state, and a new round of electrophoresis analysis can be started from the first step.

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Abstract

一种全自动高精度毛细管电泳仪，包括电泳系统、进样流路和自动取样流路；进样流路包括与四通连接器（3）连接的分流废液瓶（4）、四通微量进样阀（5）和缓冲液注射泵（1），四通微量进样阀（5）连接并向电泳系统的电泳分离毛细管（7）定量进样；自动取样流路包括取样针（15）、样品盘（16）、清洗槽（17）、试剂瓶（20）、缓冲管（14）、六端液体分配器（13）和注射器泵（19），注射器泵（19）的三端分配阀分别连接清洗液瓶（21）、自动进样器废液瓶（22）和缓冲管（14），六端液体分配器（13）的固定接口连接缓冲管（14），与固定接口轮流切换连接的六个分配接口分别连接取样针（15）、四通连接器（3）、四通微量进样阀（5）以及三个试剂瓶（20），取样针（15）切换插入清洗槽（17）或样品盘（16），自动进样器废液瓶（22）与四通微量进样阀（5）连接。上述毛细管电泳仪进样速度快，准确度高，重现性好，广泛用于不同物质的毛细管电泳自动分析。

Description

全自动高精度毛细管电泳仪 技术领域
本发明涉及物质的毛细管电泳， 具体涉及一种全自动高精度毛细管电泳仪， 属 于分析测试技术领域。
背景技术
毛细管电泳始于 20世纪 80年代。在进行分析时将毛细管内充满缓冲溶液, 毛细 管两端施加高压电 （一般 0到 +30 kV或到 -30 kV), 毛细管内的电渗流使溶液向检 测仪一端流动， 也带动毛细管中的所有物质向检测仪一端流动。 溶液内的物质由于 其电泳淌度不同， 即所带电荷与其分子量的比值（荷质比） 不同， 因此到达毛细管 终端检测仪的速度不同， 从而得到分离和检测。
毛细管电泳系统就是利用毛细管中被分析的带电物质在电场作用下移动速率不 同的原理， 达到分离的目的， 其主要部件包括一长管形毛细管、 毛细管两端的液体 容器、 电极、 高压电源供应器、 检测器和一个数据输出和处理装置。
毛细管的常规进样方式有两种： 流体力学和电迁移进样。 流体力学进样可以通 过虹吸、 在进样端加压或检测 H¾抽空等方法来实现， 电迁移进样是施加电压， 通 过电场作用， 依靠样品离子的电迁移和 （或） 电渗流将样品注入毛细管。 上述两种 进样方式存在下列缺点： 1. 由于两种方法都采用 "蘸取"的方式完成进样， 既把毛 细管蘸入样品瓶中， 施加压力或电压进样， 再移回到溶液瓶中实施分离， 进入毛细 管的样品量只能靠粗略的估算， 因此两种进样方式都缺乏准确度； 2. "蘸取"进样 时， 移动过程中毛细管端口的样品液滴大小受环境影响会发生很大变化， 使得该进 样精确度 （重复性） 很差； 3.进样时须切断施加于毛细管两端的高压电场， 使得已 建立好的电场不时地被打断再重新建立， 导致分析结果的精度不理想； 3. "蘸取" 式进样容易造成样品与缓冲液以及不同样品之间的相互污染； 4. 电迁移进样方式会 产生电歧视现象， 从而进一步降低分析的准确性和可靠性。
总之， 毛细管电泳仪的进样量非常小， 都是纳升 （nl) 级， 现有毛细管电泳仪 采用的传统进样方式无法完成如此小体积的准确定量， 毛细管端蘸取进样的方式， 其进样重复性误差大， 很难用于定量测量。
发明内容
本发明所要解决的技术问题是克服现有技术的不足， 提供一种全自动高精度毛 细管电泳仪， 实现自动定量进样， 提高进样的准确度、 重现性和可靠性； 消除电歧 视现象和样品之间的相互污染； 提高进样速度， 缩短进样时间， 最大限度地避免外 界空气进入进样流路中； 同时实现不同试剂对毛细管分离柱的自动清洗和平衡。
本发明解决其技术问题的方案如下：
一种全自动高精度毛细管电泳仪， 包括有电泳系统， 该电泳系统包括出口插入 缓冲液出液瓶的电泳分离毛细管、 依次连接在该电泳分离毛细管上的柱温箱和检测 器， 一高压电源的一极插入所述缓冲液出液瓶中， 另一极通过电隔离装置连接所述 电泳分离毛细管且在该电泳分离毛细管中形成高压电场， 其特征在于：
所述毛细管电泳仪还包括有与所述电泳系统连接的进样流路和与该进样流路连 接的自动取样流路；
所述进样流路包括与一四通连接器的三个接口分别连接的分流废液瓶、 四通微 量进样阀和缓冲液注射泵；
所述四通微量进样阀能够准确定量， 用于向所述电泳分离毛细管定量进样， 包 括有 S端口、 W端口、 P端口和 C端口共四个固定管路端口以及可旋转的内置定量 环， 该 C端口与所述电泳分离毛细管连接， 该 P端口与所述四通连接器连接， 该内 置定量环设置有一旁路通路和一具有固定容积的定量通路， 随所述内置定量环的旋 转， 所述旁路通路和定量通路在连通所述 S端口与 W端口和连通 P端口与 C端口 之间切换；
所述自动取样流路包括取样针、 清洗液瓶、 试剂瓶以及由具有定量功能的缓冲 管的两端连接起来的六端液体分配器和注射器泵；
所述注射器泵配有能够切换完成排气和清洗功能的三端分配阀， 该三端分配阀 的三个阀接口分别连接所述清洗液瓶、 自动进样器废液瓶和缓冲管；
所述六端液体分配器具有一个固定接口和能够与该固定接口轮流转换连接的六 个分配接口， 该固定接口连接所述缓冲管， 该六个分配接口分别连接所述取样针、 四通连接器的一个接口、 四通微量进样阔的 S端口以及三个装有用以清洗和平衡所 述电泳分离毛细管的不同试剂的试剂瓶；
所述清洗液瓶与自动进样器废液瓶之间连接有用以清洗所述取样针的清洗槽， 所述取样针能够切换插入该清洗槽或装有不同样品的样品盘；
所述自动进样器废液瓶与所述四通微量进样阀的 W端口连接。
作为进一步的改进， 所述的电泳系统还包括平衡废液瓶， 所述电泳分离毛细管 的出口能够切换连接该平衡废液瓶或缓冲液出液瓶。
作为进一步的改进， 所述的四通微量进样阀的定量通路的容积为 InL到 20 nL 之间的任何容积。
作为进一步的改进， 所述的缓冲液注射泵与四通连接器之间连接有用于检测管 路工作压力的毛细管压力传感器， 所述的注射器泵的三端分配阀与缓冲管之间连接 有检测取样流路工作压力的取样流路压力传感器。
作为进一步的改进， 所述的样品盘具有恒温和制冷结构。
作为进一步的改进， 所述的清洗液瓶装有清洗液， 该清洗液为乙醇或者去离子 水。
作为进一步的改进， 所述的毛细管电泳仪所有构件的功能由计算机程序实现自 动控制。
作为进一步的改进， 所述的四通连接器与分流废液瓶之间连接有用以平衡所述 电泳分离毛细管内部压力的分流管。
作为进一步的改进，所述的检测器为紫外检测器或者其他的毛细管柱上检测器。 与传统的毛细管电泳仪相比较， 本发明的自动取样流路通过一个带三端分配阀 的注射器泵和六端液体分配器联合使用， 将样品先通过大通径的管路快速抽入两者 间的缓冲管中， 然后切换六端液体分配器的通路， 将样品通过正压打入四通微量进 样阔中， 从而克服了负压进样导致的进样速度慢等缺点， 大大缩短进样时间， 同时 最大限度的避免外界空气进入进样流路中， 大幅度提高了进样的可靠性， 实现了不 同试剂对毛细管分离柱的自动清洗和平衡， 消除了电歧视现象和样品之间的相互污 染。 此外， 本发明采用了一个内置定量环的纳升级的四通微量进样阀， 其定量体积 为 InL到 20 nL之间的任一确定的容积， 通过其切换完成了精准定量进样， 本发明 的进样方式克服了传统的毛细管端蘸取或者气体压力进样方式不能完成如此小体积 准确进样的缺点， 实现了自动定量进样， 提高了进样的重现性， 进样重复性相对标 准偏差达到了 3%以下。
本发明所述全自动高精度毛细管电泳仪具有进样速度快、 定量准确度好、 精确 度高、 重现性好和易于产业化的优点， 实现了样品进样、 毛细管清洗和平衡的自动 化， 能够广泛用于不同物质的毛细管电泳分析。
附图说明
图 1是本发明的结构示意图。
图 2是本发明的工作过程原理图之一。
图 3是本发明的工作过程原理图之二。
图 4是本发明的工作过程原理图之三。 图 5是本发明的工作过程原理图之四。
图 6是本发明的工作过程原理图之五。
图 7是本发明的工作过程原理图之六。
图 8是本发明的工作过程原理图之七。
图中，
1 缓冲液注射泵， 2毛细管压力传感器， 3 四通连接器， 4分流废液瓶， 5 四通微量进样阀， 51 定量通路， 52 旁路通路， 6 电隔离装置， 7 电泳分离毛细管， 8 柱温箱， 9高压电源， 10检测器，
11 缓冲液出液瓶， 12平衡废液瓶， 13 六端液体分配器, 14缓冲管， 15取样针， 16样品盘， 17清洗槽， 18取样流路压力传感器,
19注射器泵， 20试剂瓶， 21 清洗液瓶， 22 自动进样器废液瓶， 23 分流管。
具体实施方式
下面结合附图和具体实施例对本发明作详细的说明， 但下述的实施例并非用来 限定本发明的范围， 凡依据本说明书的内容所做的等效变化及修改， 都应属于本发 明专利申请要求保护的范围。
本发明所述的全自动高精度毛细管电泳仪用于不同物质的毛细管电泳分析。 请 参阅图 1， 图示全自动高精度毛细管电泳仪包括有电泳系统、 进样流路和自动取样 流路， 该进样流路与所述电泳系统连接， 该自动取样流路与所述进样流路连接。
所述电泳系统包括电泳分离毛细管 7、 柱温箱 8、检测器 10、 高压电源 9、 电隔 离装置 6、缓冲液出液瓶 11和平衡废液瓶 12。所述电泳分离毛细管 7是物质毛细管 电泳分离的场所， 也是本系统进行分离分析的核心原件， 其出口插入所述缓冲液出 液瓶 11 ; 所述柱温箱 8对电泳分离毛细管 7进行温度控制， 主要是用以在某些电泳 分离时散出产生的焦耳热， 和加热防止毛细管电泳分离实验时毛细管内部物质的低 温结晶； 所述检测器 10用于对分离后信号的检测， 可以是紫外检测器或者其他的毛 细管柱上检测器， 该柱温箱 8和检测器 10依次连接在所述电泳分离毛细管 7上； 所 述缓冲液出液瓶 11为电泳分离分析时电泳分离毛细管 7的出口；所述高压电源 9的 一极插入所述缓冲液出液瓶 11中，另一极通过电隔离装置 6连接所述电泳分离毛细 管 7， 以在该 7 电泳分离毛细管中形成高压电场； 所述电泳分离毛细管 7的出口能 够切换连接所述平衡废液瓶 12或缓冲液出液瓶 11 ,当清洗或平衡电泳分离毛细管 7 时， 需要将电泳分离毛细管 7的出口切换到平衡废液瓶 12中， 否则会影响缓冲液出 液瓶 11中缓冲液的 pH值， 从而影响测量结果。
所述进样流路包括缓冲液注射泵 1、 毛细管压力传感器 2、 四通微量进样阀 5、 四通连接器 3和分流废液瓶 4。 所述缓冲液注射泵 1用于向所述电泳分离毛细管 7 提供补充缓冲液及一定的压力； 所述毛细管压力传感器 2用于检测所述电泳分离毛 细管 Ί和分流管路的工作压力； 所述分流废液瓶 4用于容纳通过所述四通连接器 3 分流后的缓冲液。
所述的四通微量进样阀 5具有准确定量功能， 包括有 S端口、 W端口、 P端口 和 C端口共四个固定管路端口以及可旋转的内置定量环， 该内置定量环设置有一旁 路通路 52和一定量通路 51， 该定量通路 51具有 InL到 20 nL之间的任一固定量的 容积， 如 4 nL、 10 nL或 20 nL， 以实现纳升级的定量取样； 随所述内置定量环的旋 转，所述旁路通路 52和定量通路 51在连通所述 S端口与 W端口和连通 P端口与 C 端口之间切换， 具体地说， 当在充液的位置时， 所述定量通路 51连通所述 S端口与 W端口， 同时所述旁路通路 52连通所述 P端口与 C端口， 当内置定量环旋转 180° 在注液的位置时， 所述旁路通路 52连通所述 S端口与 W端口， 而所述定量通路 51 连通所述 P端口与 C端口；所述四通微量进样阀 5的 C端口与所述电泳分离毛细管 7连接， 用于向所述电泳分离毛细管 7进行定量进样。
所述四通连接器 3的三个接口分别连接所述缓冲液注射泵 1、 分流废液瓶 4和 四通微量进样阀 5的 P端口； 所述毛细管压力传感器 2连接于所述缓冲液注射泵 1 与四通连接器 3之间， 所述四通连接器 3与四通微量进样阀 5的 P端口之间连接有 一过滤器， 用以过滤样品中和试剂中的大颗粒杂质， 保护四通微量进样阀 5和电泳 分离毛细管 7， 所述四通连接器 3与分流废液瓶 4之间连接有分流管 23， 该分流管 23通过改变口径和长度以平衡所述电泳分离毛细管 7的内部压力。
所述自动取样流路包括六端液体分配器 13、取样针 15、样品盘 16、清洗槽 17、 缓冲管 14、 试剂瓶 20、 取样流路压力传感器 18、 注射器泵 19、 清洗液瓶 21和自动 进样器废液瓶 22。
所述六端液体分配器 13具有一个固定接口和能够与该固定接口轮流转换连接 的六个分配接口； 该固定接口连接所述缓冲管 14， 该六个分配接口分别连接所述取 样针 15、 四通连接器 3的一个接口、 四通微量进样阀 5的 S端口以及三个装有用以 清洗和平衡所述电泳分离毛细管 7的不同试剂的试剂瓶 20。
所述注射器泵 19为整个系统流路动力的来源，其配有能够切换完成系统排气和 清洗功能的三端分配阀， 该三端分配阀的三个阀接口分别连接所述清洗液瓶 21、 自 动进样器废液瓶 22和缓冲管 14。 所述取样流路压力传感器 18用于检测取样流路的 工作压力， 其连接于所述注射器泵 19的三端分配阀与缓冲管 14之间。
所述清洗液瓶 21装有清洗液， 该清洗液也是进样流路的流动相， 一般选用乙醇 或者去离子水。所述自动进样器废液瓶 22用于容纳系统清洗后的废液， 其与所述四 通微量进样阀 5的 W端口连接。所述清洗槽 17用以同时完成所述取样针 15的外壁 和内壁的清洗， 其连接于所述清洗液瓶 21与自动进样器废液瓶 22之间。
所述缓冲管 14具有一定的定量功能，为系统中临时存放包括样品、进样流动相、 试剂、 清洗液等液体的处所； 该缓冲管 14 的两端将所述六端液体分配器 13和注射 器泵 19连接起来， 通过该组合构件的协同工作， 可以完成系统排气、 清洗 (平衡)、 自动定量进样等功能。
所述样品盘 16装有不同的样品， 其具有恒温和制冷结构， 能使样品保持在很低 的温度， 保持生物酶的活性， 减少样品的挥发； 所述取样针 15具有穿刺功能， 能够 穿过样品瓶的橡皮瓶塞深入到样品瓶内部取样，该取样针 15能够切换插入所述清洗 槽 17或样品盘 16。
所述毛细管电泳仪所有构件的功能都由计算机程序实现自动控制， 从而达到本 发明的全自动运行。
本发明所述全自动高精度毛细管电泳仪的工作原理如下：
第一步，参见图 2，所述电泳分离毛细管 7的出口端切换至缓冲液出液瓶 11中， 所述四通微量进样阀 5位于充液的位置， 即所述四通微量进样阀 5中的定量通路 51 连通所述 S端口与 W端口； 所述六端液体分配器 13转换连通取样针 15， 该取样针 15插入样品盘 16中， 所述注射器泵 19吸入"置换体积"的样品 (一般不小于 1.5倍取 样针 15的体积)， 使之充满取样针 15， 然后再继续抽入一定量的空气， 使样品完全 进入所述缓冲管 14中; 从而用样品冲洗取样针 15的内壁， 置换掉管路中的清洗液 及干扰组分， 使之不会注入到分离流路中。
第二步，参见图 3，所述六端液体分配器 13转换连通四通微量进样阔 5的 S端， 注射器泵 19推出"置换体积"的样品进入四通微量进样阀 5的内置定量环中，使样品 充满所述定量通路 51达到定量， 同时样品冲洗四通微量进样阀 5的内壁， 将管路中 的清洗液及干扰组分推出四通微量进样阀 5的 W端流入所述自动进样器废液瓶 22， 而不会注入到分离流路中。
第三步， 参见图 4， 所述内置定量环旋转 180°， 四通微量进样阀 5切换到注液 的位置， 即四通微量进样阀 5中充满样品的定量通路 51连通所述 P端口与 C端口， 与所述电泳分离毛细管 7联通；所述缓冲液注射泵 1的压力使定量通路 51中的样品 进入电泳分离毛细管 7， 开始进行样品的定量电泳分析； 同时， 所述取样针 15移动 至清洗槽 17中。
第四步， 参见图 5， 待定量通路 51中的样品被全部推出后， 所述内置定量环旋 转 180°, 四通微量进样阀 5切换回到充液的位置， 即所述定量通路 51连通所述 S 端口与 W端口； 在进行毛细管电泳分析的同时， 所述六端液体分配器 13保持与四 通微量进样阀 5 的 S端连通， 注射器泵 19将清洗液瓶 21内的清洗液注入四通微量 进样阀 5， 以对其内置定量环进行小流量清洗。
第五步， 参见图 6， 在进行毛细管电泳分析的同时， 所述六端液体分配器 13转 换连通插入所述清洗槽 17的取样针 15, 所述注射器泵 19将清洗液注入取样针 15 以对取样针 15及取样流路进行清洗。
第六步， 参见图 7, 毛细管电泳分析完成后， 进入试剂清洗与平衡电泳分离毛 细管 7阶段； 所述六端液体分配器 13转换连通所需要的试剂瓶 20， 用注射器泵 19 将清洗和平衡电泳分离毛细管 7用的试剂吸入缓冲管 14中。
第七步， 参见图 8， 所述电泳分离毛细管 7的出口端切换至平衡废液瓶 12， 所 述六端液体分配器 13转换连通四通连接器 3， 用所述注射器泵 19以特定的速度将 缓冲管 14中的试剂通过所述四通微量进样阀 5的旁路通路 52推入电泳分离毛细管 7, 对电泳分离毛细管 7进行清洗和平衡， 清洗后的废液进入所述平衡废液瓶 12。
第八步， 仍见图 8, 所述六端液体分配器 13保持与四通连接器 3相通， 用注射 器泵 19将所述清洗液瓶 21中的清洗液注入缓冲管 14，进而注入电泳分离毛细管 7, 从而对整个缓冲管 14和电泳分离毛细管 7进行清洗。
完成清洗后， 所述全自动高精度毛细管电泳仪还原初始状态， 可以从第一步开 始新一轮的电泳分析。
在上述工作过程中， 所述毛细管电泳仪所有构件的功能都由计算机程序实现自 动控制， 从而达到全自动运行。
本发明要求的保护范围不仅限于上述实施例， 也应包括其他显而易见的变换和 替代方案。

Claims

权利 要 求
1、一种全自动高精度毛细管电泳仪， 包括有电泳系统， 该电泳系统包括出口插 入缓冲液出液瓶的电泳分离毛细管、 依次连接在该电泳分离毛细管上的柱温箱和检 测器， 一高压电源的一极插入所述缓冲液出液瓶中， 另一极通过电隔离装置连接所 述电泳分离毛细管且在该电泳分离毛细管中形成高压电场， 其特征在于：
所述毛细管电泳仪还包括有与所述电泳系统连接的进样流路和与该进样流路连 接的自动取样流路；
所述进样流路包括与一四通连接器的三个接口分别连接的分流废液瓶、 四通微 量进样阀和缓冲液注射泵；
所述四通微量进样阀能够准确定量， 用于向所述电泳分离毛细管定量进样， 包 括有 S端口、 W端口、 P端口和 C端口共四个固定管路端口以及可旋转的内置定量 环， 该 C端口与所述电泳分离毛细管连接， 该 P端口与所述四通连接器连接， 该内 置定量环设置有一旁路通路和一具有固定容积的定量通路， 随所述内置定量环的旋 转， 所述旁路通路和定量通路在连通所述 S端口与 W端口和连通 P端口与 C端口 之间切换；
所述自动取样流路包括取样针、 清洗液瓶、 试剂瓶以及由具有定量功能的缓冲 管的两端连接起来的六端液体分配器和注射器泵；
所述注射器泵配有能够切换完成排气和清洗功能的三端分配阀， 该三端分配阀 的三个阀接口分别连接所述清洗液瓶、 自动进样器废液瓶和缓冲管；
所述六端液体分配器具有一个固定接口和能够与该固定接口轮流转换连接的六 个分配接口， 该固定接口连接所述缓冲管， 该六个分配接口分别连接所述取样针、 四通连接器的一个接口、 四通微量进样阀的 s端口以及三个装有用以清洗和平衡所 述电泳分离毛细管的不同试剂的试剂瓶；
所述清洗液瓶与自动迸样器废液瓶之间连接有用以清洗所述取样针的清洗槽， 所述取样针能够切换插入该清洗槽或装有不同样品的样品盘；
所述自动进样器废液瓶与所述四通微量进样闽的 w端口连接。
2、 如权利要求 1所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的电泳 系统还包括平衡废液瓶， 所述电泳分离毛细管的出口能够切换连接该平衡废液瓶或 缓冲液出液瓶。
3、 如权利要求 1或 2所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的 四通微量进样阀的定量通路的容积为 InL到 20 nL之间的任何容积。
4、如权利要求 1所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的缓冲 液注射泵与四通连接器之间连接有用于检测管路工作压力的毛细管压力传感器， 所 述的注射器泵的三端分配阀与缓冲管之间连接有检测取样流路工作压力的取样流路 压力传感器。
5、如权利要求 1所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的样品 盘具有恒温和制冷结构。
6、如权利要求 1所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的清洗 液瓶装有清洗液， 该清洗液为乙醇或者去离子水。
7、如权利要求 1所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的毛细 管电泳仪所有构件的功能由计算机程序实现自动控制。
8、如权力要求 1所述的全自动高精度毛细管电泳仪， 其特征在于： 所述的四通 连接器与分流废液瓶之间连接有用以平衡所述电泳分离毛细管内部压力的分流管。
9、如权力要求 1所述的全自动髙精度毛细管电泳仪， 其特征在于： 所述的检测 器为紫外检测器或者其他的毛细管柱上检测器。
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