CN1068944C - Counter-flow focusing electrophoresis apparatus - Google Patents
Counter-flow focusing electrophoresis apparatus Download PDFInfo
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- CN1068944C CN1068944C CN 98104828 CN98104828A CN1068944C CN 1068944 C CN1068944 C CN 1068944C CN 98104828 CN98104828 CN 98104828 CN 98104828 A CN98104828 A CN 98104828A CN 1068944 C CN1068944 C CN 1068944C
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Abstract
The present invention relates to a counter-flow focusing electrophoresis device and method for separating and analyzing a (biological) chemical system. The device is provided with an electrophoretic channel filled with electrophoretic solutions, a solid-phase ionic conductor which is mutually in contact with the channel in parallel, an electrophoretic solution driver, a positive electrode chamber, a negative electrode chamber, a positive electrode, an electrode negative and a direct current power supply. Both ends of the channel are respectively connected with the electrophoretic solution driver and the electrode chambers, the ratio V<x>/epsilon<x> of the linear velocity V<x> of the electrophoretic solutions to the electric field strength epsilon<x> of the panting of ions to a x direction driven by the electrophoretic solution is monotonic quantity of the x. The present invention has the advantages of unique focusing principle, simultaneous realization of separation, concentration and focusing, short electrophoretic channel, low service voltage, high analyzing speed and wide range of application.
Description
The present invention relates to a kind of apparatus and method of separating and analyze (biology) chemical system with electrophoresis.
In recent ten years, the electrophoretic separation analytical technology is developed rapidly, it utilizes different ions (charged particle) to have different movement velocitys and form different Zone electophoresis bands in electric field, according to different disjunctive models and separating property, can be divided into zone electrophoresis, isotachophoresis and isoelectric focusing electrophoresis, especially in recent years, the document sharp increase of relevant capillary zone electrophoresis, each big instrument company tries to be the first and releases the instrument of various models since 1989, but in order to obtain high resolution and velocity of separation, capillary zone electrophoresis must apply the high pressure of tens thousand of volts at the separator tube two ends, sample size can only be received upgrading for Na Kai even Asia, and want accurately and to inject kapillary all be a very big difficult problem receiving the sample of upgrading on robotization ground, and the very little detectable concentration lower limit that also causes of sample size is undesirable, in order to improve the lower limit of detectable concentration, must find can the on-the-spot desired solution that concentrates; The sample area band is in capillary motion and shift to detection window gradually, makes the development of array Capillary Electrophoresis run into the too complicated difficulty of detection system.
Purpose of the present invention aim to provide a kind of on the principle essence ground change existing various electrophoretic analysis patterns, the realization sample separation is located focusing by the size of its effective electromobility, have district's band and concentrate focusing automatically, peak capacity is big, operating voltage is low, electrophoresis path is short, and analysis speed is fast, the counter-flow focusing electrophoresis apparatus of wide accommodation.
The present invention is provided with the electrophoresis path that is full of electrophoresis solution, and the length direction coordinate is x; Be provided with solid phase ionic conductor parallel with electrophoresis path and that be in contact with one another in passage, its length direction is identical with x: be provided with the electrophoresis solution driver, the two ends of electrophoresis solution driver are connected with the electrophoresis path two ends.The electrophoresis solution that drives electrophoresis path flows in the x direction, flow direction with treat measured ion swimming side same opposite (adverse current), its linear velocity v
xWith electrophoresis solution drive ion towards x side with the electric field intensity ε of swimming
xRatio v
x/ ε
xMonotonic quantity for x.Be provided with the positive and negative electrode chamber.The positive and negative electrode chamber connects the two ends of electrophoresis path respectively, and positive and negative electrode is set, and positive and negative electrode connects direct supply.Electrolyte solution is injected in the positive and negative electrode chamber: after the energising, and electric field ε
xDrive negative ion to be measured to positive electrode direction swimming (to separate negative ion is example, down together).
Said electrophoresis path is the liquid phase electric conductor, for example, is full of electrophoresis solution pipeline, raceway groove or utilizes capillarity to be filled the porous medium (as paper slip, gel or cellulose membrane) of electrophoresis solution.
Said solid phase ionic conductor can be banded, for example Zeo-karb band or ionomer polymer strip.
The solid phase ionic conductor closely contacts with electrophoresis path, becomes parallel electric conductor, and the kation between two electric conductors can be crossed two intersection interfaces and flow.
Counter-flow focusing electrophoresis is a kind of electrophoresis new technology that is different from zone electrophoresis, isotachophoresis, isoelectric focusing, and it has district's band and concentrates distinguishing features such as focusing, peak capacity is big, operating voltage is low automatically.Just as isoelectric focusing; Fast and the applicable object of analysis speed extensively then as zone electrophoresis.Can be used for the compartment analysis of inorganics and organism (large and small molecule), all have wide practical use in fields such as Biochemical Research, clinical examination and food, medicine, environmental protection.
Adverse current of the present invention is focused on (Counter Flow Focusing; Be called for short CFF), i ion to be separated is done directed movement at electrophoresis path (x direction), under the flow of solution state, its directed movement net velocity v
IxBe v
Ix=v
x-ε
xμ
Ix, v wherein
xThe linear velocity that flows in the x direction for electrophoresis solution; ε
xFor driving the electric field intensity of ion towards the swimming of x direction; μ
IxBe the effective electromobility of ion at the x place.In different electrophoresis types, v
IxDifferent Results is arranged.
For isoelectric focusing (IEF), electrophoresis solution (or gel) does not flow, so v
x=0, and employed electrophoresis solution is an ampholyte solution, its pH is x function (forming the pH gradient), cause ampholyte μ
IxBe the function of x, at the μ of isoelectric point place
x=0, so v
Ix=0 (can focus on), this method be only applicable to protein and so on ampholyte separation.
For zone electrophoresis (ZE), ε
x, μ
IxBe irrelevant constant, so v with x
IxIrrelevant with x, but relevant with ionic species i, each district's band can not focus on before the different speed constant speed and then finally to finish branch high.Because of diffusion, convection current etc. are former thereby prolong and cause dilution in advancing for district band.
For isotachophoresis (ITP), after separating, regulates by district's band each district's band concentration and electric field automatically, make to be inversely proportional to this ion mobility each component district band ε
xμ
IxProduct constant, thereby v
IxAll irrelevant with component and x.Each district's band is connected and constant speed is advanced, and can not focus on.
And the present invention (counter-flow focusing electrophoresis need not form the pH gradient, μ in CFF)
IxThough be constant, the technical electrophoresis path that utilizes above-mentioned particular design has been realized v
x/ ε
xBe the x monotonic quantity, as v/ ε
x=μ
iThe place, v
Ix=0, so the i ion can focus on x place, corresponding position.Separate the concentrated three of focusing and realize that simultaneously the detectable concentration lower limit reduces, electrophoresis path shortens dramatically, and operating voltage greatly descends, and helps realizing channel arrayization.In addition, owing to need not gradient pH damping fluid, as long as effective electromobility μ
iVariant promptly separable, thus be not limited to protein and so on ampholyte separation, have wide range of applications.
The comparison of the present invention and existing three major types electrophoretic techniques can see the following form:
| The electrophoretic techniques title | Characteristics and function | Separate foundation and range of application | Can on-the-spotly concentrate, focus on? | |
| Present technique | Counter-flow focusing electrophoresis (CFF) | Flow of solution and electromigration are reverse, and specific channel designs, and make the net velocity v of each ion IxRelevant with the position.Each ion can focus on its net velocity v respectively IxNull position. | Separate according to the effective ion mobility difference.Range of application and zone electrophoresis are extensive equally. | Focus on and concentrate.Electrophoresis path is short.Operating voltage is low. |
| Prior art | Isoelectric focusing electrophoresis (IEF) | Special buffer medium makes pH relevant with the position.Energy isolated protein and so on, and focus on the pH position of isoelectric point separately. | Separate according to isoelectric point difference; Can only be used for analysing protein and so on. | Focus on and concentrate, electrophoresis path is short. |
| Isotachophoresis (ITP) | Automatically regulate and respectively distinguish charged, make to be inversely proportional to this ion mobility, each district's band is connected and constant speed is advanced.Can not focus on, peak capacity is not very big. | Separate according to the effective ion mobility difference. | Out-focus may concentrate or thinning.Electrophoresis path is long. | |
| Zone electrophoresis (ZE) | PH and electric field all and location independent, each ion district band with different speed constant speed before so that finally finish separation, can not focus on. | Separate according to the effective ion mobility difference.Have wide range of applications. | Out-focus does not concentrate, and is thinning in time.Electrophoresis path is long. | |
In sum, the present invention has following advantage:
1. from inspissation: focus on the back sample component and be limited within extremely short district's band, concentration can improve an order of magnitude, and it is identical with isoelectric focusing, and zone electrophoresis does not possess this effect, though isotachophoresis has certain inspissation; But district's band concentration of component can not be improved by leading electrolyte solution control, because its this effect, the peak capacity of counter-flow focusing electrophoresis is big.
2. district band is from sharpening: when the i ion departs from focus point because of diffusion or convection current, can get back to focus point automatically under electric field intensity and two kinds of factors of reverse current act on simultaneously.
3. electrophoresis path is lacked (being generally several centimetres), operating voltage low (being generally hundreds of volts), thereby disengaging time is short, and thermal value is little, and the instrument Miniaturizable is cheap, safe in utilization.
4. behind focus state, district's band can be stablized constant (not moving) for a long time, is convenient to monitor separating overall process; Passage is short and straight, helps multichannel array type, can adopt CCD to videotape technology.
Fig. 1 is a structural representation of the present invention.
Fig. 2 is the structural representation of two electric conductors of solid-liquid.
Fig. 3 separates and focusing effect for bromjophenol blue and amino black.
The present invention is described further below in conjunction with example.
As shown in Figure 1, the present invention includes electrophoresis path and solid phase ionic conductor (1), electrophoresis solution driver (2), electrode chamber (3,4), positive and negative platinum electrode (5,6) and direct supply (7) parallel with electrophoresis path and that in passage, be in contact with one another.For being full of the passage of electrophoresis solution, become the liquid phase electric conductor referring to Fig. 2 electrophoresis path (L), the length direction coordinate is x; Parallel with electrophoresis path (L) and its length direction of solid phase ionic conductor (S) that be in contact with one another at whole passage is identical with x.Common kation (Na is for example arranged in two electric conductors of solid-liquid movably
+), and can cross two-phase interface and flow, negative ion to be measured then denies admission to the solid phase ionic conductor.As one of embodiment, solid phase cationic electroconductive body can be selected a kind of Zeo-karb material for use.
The solution of electrophoresis path is subjected to the common control of the fluid pressure differential at electric osmose power and passage two ends and flows that (linear velocity is v
x), and the ion flow in the drive solution.Flow direction opposite with the swimming for the treatment of measured ion (electromigration) direction (adverse current is utilized electrophoresis solution driver for example pump or hydraulic pressure difference formation system etc.).In order to realize the condition of adverse current focusing principle requirement, i.e. v
x/ ε
xBe the x monotonic quantity, multiple scheme can be arranged.As one of embodiment, can adopt electrophoresis path (L is referring to Fig. 2) constant cross-section, so the adverse current linear velocity v of any position of whole passage
xAll constant with the liquid phase conductance, and irrelevant with x; And be the monotonic quantity of x coordinate as the xsect (or conductance) of the solid phase ionic conductor of one of parallel conductance body, obviously, the x direction forms two electric conductor parallel connections during energising, so when energising, the electric field ε that the x direction forms
xBe the function of x, this has just realized v
x/ ε
xSatisfied the condition of adverse current focusing principle requirement for the x monotonic quantity.
If a certainly treat that effective electromobility of measured ion i is μ
i, at a certain position of the passage v of x place
x/ ε
x=μ
I, thereby the net velocity of i ion is zero (v
Ix=v
x-ε
xμ
i=0), thus i ion focus at the x place.μ
iIon inequality then focuses on x place inequality.Reach to separate to concentrate and focus on the effect that the three realizes synchronously.
The material of electrophoresis path can adopt the chemical inertness insulating material, quartz material for example, and glass, plastics etc., passage length are 1-10cm, the cross section is 10
-7-10
-2Cm
2, be full of damping fluid.The only hundreds of volts of the direct supply voltage that applies, for example 100-1000 volt.After applying direct supply voltage and controlling the solution adverse current, treat respectively that measured ion promptly progressively is separated from each other and according to its effective electromobility μ
iSize on different positions, focus on.It is that bromjophenol blue and amino black potpourri (respectively are 7 * 10 that Fig. 3 provides sample
-7Mol/l, annotating the sample volume is 2 * 10
-3Cm
3) separation and focusing effect, the original state that curve a, b, c represent respectively to separate, intermediateness and final focus state.
Detection method of the present invention and means can utilize methods such as the existing various optics that are used for various electrophoresis, galvanochemistry, mass-spectrometry and means to carry out.
Claims (4)
1, counter-flow focusing electrophoresis apparatus, it is characterized in that this device has an electrophoresis path, by the electrophoresis path/in solid phase ionic conductor parallel with electrophoresis path and that be in contact with one another in passage is arranged, electrophoresis path links to each other with the electrophoresis solution driver, respectively there is an electrode chamber at the two ends of electrophoresis path, electrode is housed in the electrode chamber and is filled with or is full of electrolyte solution, electrode links to each other with direct supply, electrophoresis solution flows in the electrophoresis solution driver drives electrophoresis path, flow direction with treat that measured ion swimming direction is opposite, its linear velocity v
xWith electric field intensity ε
xRatio v
x/ ε
xMonotonic quantity for electrophoresis path length direction coordinate x, said electrophoresis path constant cross-section, the interfacial area of solid phase ionic conductor or conductance are the monotonic quantity of electrophoresis path length direction x coordinate, electrophoresis path adopts the chemical inertness insulating material, passage length is 1～10cm, and sectional area is 10
-7～10
-2Cm
2, direct supply voltage is 100～1000 volts.
2, counter-flow focusing electrophoresis apparatus as claimed in claim 1 is characterized in that said electrophoresis path is the pipeline that is full of electrophoresis solution, the porous medium of raceway groove or energy imbibition.
3, counter-flow focusing electrophoresis apparatus as claimed in claim 2 is characterized in that the porous medium of said energy imbibition is paper slip, gel or cellulose membrane.
4, counter-flow focusing electrophoresis apparatus as claimed in claim 1 is characterized in that said solid ion conductive body can be ion exchange resin band or ionomer polymer strip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98104828 CN1068944C (en) | 1998-01-15 | 1998-01-15 | Counter-flow focusing electrophoresis apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 98104828 CN1068944C (en) | 1998-01-15 | 1998-01-15 | Counter-flow focusing electrophoresis apparatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1193107A CN1193107A (en) | 1998-09-16 |
| CN1068944C true CN1068944C (en) | 2001-07-25 |
Family
ID=5218493
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 98104828 Expired - Fee Related CN1068944C (en) | 1998-01-15 | 1998-01-15 | Counter-flow focusing electrophoresis apparatus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1068944C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5984080B2 (en) * | 2011-12-22 | 2016-09-06 | シャープ株式会社 | Control method, control device, control system, and control program |
-
1998
- 1998-01-15 CN CN 98104828 patent/CN1068944C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1193107A (en) | 1998-09-16 |
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