CN1056261A - Multifunctional free-electrophoresis technology - Google Patents

Abstract

The present invention relates to a kind of multifunctional free-electrophoresis technology, comprise three, first term is the required device of multifunctional free-electrophoresis technology.Second is to finish the required method of this multifunctional free-electrophoresis technology.The 3rd is to realize in the needed a kind of equivalent of above-mentioned technology and detection technique.Feature of the present invention is isoelectric point (PI) difference according to amphiprotic substance.The isoelectric point of amphiprotic substance being separated, concentrates, dialyses, estimates to measure amphiprotic substance by the pH value that changes buffer solution.The present invention is not only applicable to material separation on a large scale such as amino acid, polypeptide, protein, nucleic acid, purifying, concentrates, dialyses, and is applicable to a small amount of or micro-separation and purification such as above-mentioned substance even virus yet, and the estimation that concentrates the dialysis isoelectric point is measured.

Description

Multifunctional free-electrophoresis technology

The present invention is three inventions of a total inventive concept, first is the required device of multifunctional free-electrophoresis technology, second is to finish the required method of this free-electrophoresis technology, and the 3rd is to finish in the needed a kind of equivalent of above-mentioned technology and detection technique.The present invention is applicable to that multiple amphiprotic substance resembles the separation of materials such as amino acid, polypeptide, protein, nucleic acid even virus, purifying, analysis, concentrates the estimation and the mensuration of dialysis and isoelectric point, and is expected to be applied in other colloid chemistry.

This specification is described in detail and illustrates that they are background technology, device, principle methodological function, use, pluses and minuses comparison to the present invention with regard to following problem.(attached list of references in addition)

Background technology

Electrophoresis (Electrophoresis): electrophoresis be it is found that as far back as the initial stage in 19th century, but up to Tiselius after electrophoresis apparatus Fang Xiang obtains significant achievement, electrophoresis is widely used gradually as a kind of compartment analysis means.Weiland in 1948 etc. have developed with the electrophoresis method of paper as holder, electrophoretic techniques has been got back and has been used further and develop, up to the present, the electrophoretic techniques kind is a lot, but two big classes have added up to, wherein a class is no support electrophoretic techniques, and another kind of is the support electrophoretic techniques, and concrete condition is as follows:

No support electrophoresis (free electrophoresis) technology:

Tiselius electrophoresis (1)

Isoelectric point is assembled electrophoresis (2.3.4.8.16.17)

The density gradient isoelectric point is assembled electrophoresis (4)

Isotachophoresis technology (4.17)

The support electrophoretic techniques is arranged:

Filter paper and other fiber membrane electrophoresis:

Paper electrophoresis (4.17)

High pressure paper electrophoresis (4.17)

Cellulose acetate membrane electrophoresis (4)

Glass fibre film electrophoresis (4)

Gel electrophoresis technology:

Starch-gel electrophoresis (4.5)

Agargel electrophoresis (4.7)

Agarose gel electrophoresis (4)

Counter immunoelectrophoresis (16)

Rocket electrophoresis (16)

The gel electrophoresis of polypropylene enamine

Disc electrophoresis (8.16.17)

Vertical slab electrophoresis (8)

Miniature circular disc electrophoresis (9)

SDS-disc electrophoresis (8)

SDS-vertical slab electrophoresis (8)

Miniature aperture gradient electrophoresis (9)

The gel isoelectric point is assembled electrophoresis (8.17)

Miniature SDS-bore diameter gradient electrophoresis (9)

Minigel isoelectric focusing electropho-resis (9)

The powder electrophoretic techniques:

Starch electrophoresis (11)

Cellulose powder electrophoresis (12)

Glass dust electrophoresis (4.8)

Resin-oatmeal electrophoresis (4)

The filament electrophoretic techniques:

Nylon yarn electrophoresis (10)

Artificial silk electrophoresis (10)

Bidirectional electrophoresis technique:

O ' Farrell ' s electrophoresis (13)

COE (4)

The mixed gel electrophoretic techniques

Agarose-polyacrylamide gel electrophoresis (14.15)

2, gel filtration (Gel chromatography): (16) are a kind of fractionation techniques that grows up the sixties.The principle of its separation is that the gel object itself has the molecular sieve effect, when being used to separate, as sieving, big molecule and small-molecule substance is separated.Be applicable to protein, the separation of big molecule (or macromolecule) organic substance such as polysaccharide.

3, ion exchange technique (Ion exchange chromatograpny):

(4.16.17) ion exchange technique is a kind of isolation technics that grows up gradually the thirties, and its principle is:

Exch in the formula ^-For having the cation-exchanger of electric charge.X ⁺, YH ⁺, Z ⁺Be cation.Neutral molecule and anion do not combine with exchanger fully.At exchanger with after the ion of opposite charges combines as static, will with ion and uncharged molecules wash-out from separate matter of exchanger band identical charges.The mode of wash-out has two kinds, and a kind of is to increase X ⁺Concentration, make X ⁺Replace YH ⁺And Z ⁺, another kind is to increase PH, thereby makes YH ⁺And Z ⁺Change YH into ⁰And ZOH, thereby make YH ⁺And Z ⁺Be able to wash-out.Ion exchange technique, is used for separating purification of protein, polypeptide, nucleic acid and enzyme etc. as a kind of separation means aspect biochemistry.

4, the technology of saltouing of protein (Solt fractional techniques of proeins) ⁽¹⁶⁾: its principle is to add soluble-salt in protein solution, destroys the stability of protein colloid, and makes protein precipitation.Protein of different nature can be precipitated out from the protein colloid respectively by the neutral salt that adds variable concentrations, thereby reaches the purpose of separation.

5, affinity chromatography technology (Affinity chromatography ^(4.16.17): its principle is to separate and purify according to the peculiar biologically active difference of boiomacromolecules such as antigen-antibody.This separating bio polymer substance has very high purity, and generally as long as disposable purifying.

6, ultracentrifugation technology (The techniques of utracentrifugation): ⁽¹⁷⁾Be to produce 500.000xg(75.000rpm.r=8cm when utilizing high speed rotating) centrifugal force separate, analyze the method for materials such as subcellular structure, nucleic acid and protein.

Except that above-mentioned, other methods of separating, analyzing amphiprotic substance and polymer substance are also a lot.For example, adsorption chromatography ^(16.17)Thin-layer chromatography ⁽¹⁷⁾CCD ⁽¹⁷⁾Deng.Because electronic computer separates in conjunction with having produced automation with other isolation technics (as ion-exchange), the method for materials such as purifying and analysis boiomacromolecule, but these equipment are all very valuable.

The purpose of this invention is to provide a kind of free-electrophoresis technology, it can be easily according to the isoelectric point difference of amphiprotic substance, by changing the pH value of the buffer solution of two grooves up and down, amphiprotic substance is carried out separation and purification and analysis, estimate to measure the isoelectric point of amphiprotic substance, and can rarer amphiprotic substance solution be concentrated, concentrate the back dialysis.This technology is not only applicable to amphiprotic substance separates and purifying on a large scale, and is applicable to the separation and purification and the analysis of a small amount of or micro-example.Below at first describe the present invention and illustrate in conjunction with three kinds of devices.

Device

The present invention has three kinds of contrive equipments, and first kind is that the multifunctional free-electrophoresis device electrophoresis of holding concurrently concentrates dialysis apparatus, and second kind is the multifunctional free-electrophoresis device of band cooling system.The third is in the equivalent and checkout gear, below is described in detail according to the order of sequence and illustrates.

The multifunctional free-electrophoresis device electrophoresis of holding concurrently concentrates dialysis apparatus.

Fig. 1. be the three-dimensional generalized section of this device.

Fig. 2 a and Fig. 2 b are respectively profile and the front views of the electrophoresis Guan Ji in this device.

With reference to Fig. 1, as seen going up groove (1) is the container of splendid attire buffer solution, topmost is cover plate (a) at it, and electrode hole (b) and inert electrode (c) are arranged on cover plate (a), and cover plate (a) can be gone up breakdown from last groove (1).On last groove (1) downside wall discharge opeing duct (e) is arranged, discharge opeing duct (e) can connect a band tubing, during electrophoresis hose clamp closed, can be when electrophoresis finishes with the flexible pipe breakdown, and the buffer solution of last groove (1) is discharged from discharge opeing duct (e).Sidewall at last groove (1) also has a pipe clamp (g).The electrophoresis duct (d) that distribution rule is arranged in the bottom of last groove.The electrophoresis Guan Ji (4) of its main and following connection and the size in the electrophoresis duct (d) on the pressing plate (2) are consistent with distribution.Last groove (1) lower, outer perimeter have 4 markings (f) of distribution rule, the figure of marking (f) be " ".Circular stable sliding recessed (i) is arranged in the bottom surface of last groove (1), just in time can coincide closely with its ring-type stable sliding protruding (h) above electrophoresis Guan Ji (4) that is connected down.

In conjunction with Fig. 1 and Fig. 2 b, visible electrophoresis Guan Ji (4) goes up the electrophoresis duct (d) and the marking (f) of distribution rule, also has circular stable sliding protruding (h) and stable sliding recessed (i); Wherein marking (f) has two kinds of forms, and adjacent and 45 degree of being separated by of these two kinds of forms are arranged, the marking on the same groove of a kind of figure (l) of marking (f), another kind be " "; when the marking (f) on the upper and lower two adjacent electrophoresis Guan Ji (4) corresponding when identical; represent that the electrophoresis duct (d) on the upper and lower two adjacent electrophoresis Guan Ji (4) communicates fully; when mutual rotation 45 degree; make corresponding different of marking (f); represent that the electrophoresis duct (d) on upper and lower two electrophoresis Guan Ji (4) cuts off mutually fully, and be the desired positions of cutting off.Finishing above-mentioned communicating and break realizes every rotating with common axle center mutually by upper and lower two electrophoresis Guan Ji (4).

Referring to Fig. 1, with electrophoresis Guan Ji (4) that last groove (1) tightly links to each other on, discoid concave surface (j) and bolt card (k) are arranged, this concave surface (j) just in time holds penetrating film (3) and penetrating mould plate (2).Electrophoresis duct (d) is also arranged on the pressing plate (2), also have bolt mother (l), the electrophoresis duct (d) on the pressing plate (2) is consistent with size and distribution on the electrophoresis Guan Ji (4); Bolt mother (l) just in time matches with bolt card (k), and the effect that designs this office is rotation in the concave surface (j) of blocking-up pressing plate (2) on electrophoresis Guan Ji (4).

In Fig. 1, between all electrophoresis Guan Ji (4) and electrophoresis Guan Ji (4) and up and down between the groove, can rotate mutually with their common axle, and it is enough good to coincide, so that the aqueous solution can not be from these anastomotic position seepage flow.When two electrophoresis Guan Ji (4) were in the position that communicates fully up and down, the electrophoresis pipeline was formed in the electrophoresis duct (d) on all electrophoresis Guan Ji (4) in addition.

Referring to Fig. 1, groove (5) as follows.Down on the groove (5) electrode hole (b) is being arranged, inert electrode (c), electrode (c) are closely in fixed electrode hole (b).Discharge opeing duct (e) is arranged on sidewall, it acts on the discharge opeing duct (e) on the same groove (l), one exhaust annular groove (m) and exhaust regulator hole (n) are on the cover board arranged, the effect of exhaust annular groove (m) is when electrophoresis is prepared, and will descend the interior survival gas of groove (5) to discharge from exhaust annular groove (m) and electrophoresis duct (d).In following groove (5) bottom surface bearing (o) is arranged, in the groove (5) gather qi together device (6) is being arranged down, gather qi together device (6) is fixed on the bottom surface by its pin, and by exhaust regulator hole (n), its long tube is stretched out down outside the groove (1), connect a flexible pipe (7) again, flexible pipe (7) can be fixed on the pipe clamp (g) on the groove (1), the effect of this office is the gas that electrode produces when discharging electrophoresis, and the upper and lower tank liquor of balance is pressed.On the cover plate of following groove (5), a concave surface (j) is also arranged, annular stable sliding recessed (h).Penetrating film (3) is identical with the structure of uppermost electrophoresis Guan Ji (4) with pressing plate (2).

Referring to Fig. 1, when a lot of electrophoresis Guan Ji (4) fuse to one (braces is drawn together), i.e. when electrophoresis Guan Ji (4) making is very thick, can be used to very easily concentrate.

The free electrophoresis device of band cooling system:

Fig. 3 is the three-dimensional generalized section of this device.

Fig. 4 is parallel to the electrophoresis Guan Ji profile at (4) two ends in this device.

Referring to Fig. 3, as seen except that a cooling system was arranged, the structure among other structures and Fig. 1 was same or similar, at this not to give unnecessary details.This device cooling system is described in detail and illustrates below for Fig. 3 and Fig. 4.

Referring to Fig. 3, visible whole cooling system is by water knockout drum (8), and some conduits (9) and electrophoresis Guan Ji (4) form.Water knockout drum has two, and it consists of a bigger water guide duct on a backed container (is called for short big guide hole (p) and some less water guide ducts (being called for short little guide hole (q)); The effect difference of two water knockout drums, one is by conduit (9) cooling water to be sent into each electrophoresis Guan Ji (4), and another is that the cooling water that enters in each electrophoresis Guan Ji (4) is derived by conduit (9), and this arrow points from Fig. 3 mark can obviously be found out.

In conjunction with Fig. 3 and Fig. 4, visible electrophoresis Guan Ji (4) also has water inlet (r), cooling chamber (s), weather board (t) and delivery port (x) except that the structure that has in Fig. 1.The purpose of design weather board (t) is guide cooling water Rational flow in cooling chamber (S), to reach the purpose of homogenizing cooling effect.Delivery port (X) will be as far as possible in a high position, to drain the gas in cooling chamber (S) in addition.

It is to be noted that used material will have enough mechanical strengths and rigidity in Fig. 1, Fig. 3, water white transparency, can acid and alkali-resistance, relative chemical stability be arranged and will amphiprotic substance not had tangible suction-operated, this class material resembles the unorganic glass of some kind and lucite etc.Nature does not comprise soft conduit, electrode and penetrating film to the requirement of this material.

In addition, in to material requirements, it is that amphiprotic substance to require separation does not have permeability that penetrating film requires, and very little molecule or ion are resembled H ₂O, H ⁺, OH ^-Freely pass through Deng energy.This class material resembles pellicle, dialysis membrane, plays filter membrane and nuclear pore membrane etc.Requirement to electrode material also is inertia naturally, and this class material resembles platinum material etc.For soft conduit, can adopt elastomeric material, also can adopt silica gel material.

In the equivalent and checkout gear:

Fig. 5 is the structural representation of this device.

Referring to Fig. 5, as seen go up groove (1), be the container of splendid attire buffer solution.Inert electrode (2) is arranged on last groove (1), and the exhaust balance pipe (3) of upper end.Be connected to scale observation tube (4) bottom under the last groove (1) one piston (5) is arranged, the interior caliber of piston (5) is with observing the same thickness of (4) interior caliber, groove (6) under the termination under the observation tube is having inert electrode (2) air guide balance pipe (3) and bearing (7) on the groove (6) down.The gas that two air guide balance pipes (3) produce in the time of can deriving electrolysis, again can balance the pressure of two grooves up and down, two air guide balance pipes (3) internal diameter requires the same thickness, to prevent the influence of surface tension to observed result.Nature can design a cooling device (9) in observation tube (4) periphery, produces too high temperature when avoiding electrophoresis the result is exerted an influence.

Whole device is except that inert electrode, and used material requirements water white transparency acid and alkali-resistance has certain mechanical strength, and relative chemical inertness is arranged, and this class material resembles the unorganic glass of some kind and lucite etc.

Principle, method, function

Fig. 6, Fig. 7, Fig. 8, Fig. 9, Figure 10 are principle schematic of the present invention.

In Fig. 6, demonstrate groove (also claiming alkaline groove), electrophoresis pipe and following groove (also claiming acid tank); Groove all links to each other with the electrophoresis pipe across penetrating film up and down, and last groove connects cathode electrode, and following groove connects anode electrode.When the access failure dc source, the buffer solution of last groove and electrophoresis pipe top dress alkalescence is (with OH ^-And to establish pH value be PH expression), _AlkaliElectrophoresis pipe middle part dress amphiprotic substance solution resembles viper venom; Following groove and electrophoresis pipe bottom dress acid solution are (with H ⁺And to establish pH value be PH expression), _Acid, above buffer solution is rarer, and the pH value of going up the buffer solution of groove as required is not more than 11, and the pH value of acid tank buffer solution is not less than 3.

After connecting power supply, each zwitterion in the buffer solution all moves to its opposite electrode in groove and the electrophoresis pipe up and down, same OH ^-And H ⁺Also will under electric field action, produce migration.If do not have amphoteric admixture matter at electrophoresis pipe middle part, Qian Yi result will make OH so ^-And H ⁺Meet near electrophoresis pipe middle part and neutralization mutually, after connecting power supply, the electrophoresis pipe divides from pH value from top to bottom 3 district's bands will occur like this, and promptly the top pH value is PH _AlkaliThe alkalescence band, pH value is that neutral zone and the bottom pH value that pH value descends is the acid band of PH from top to bottom.As shown in Figure 7, clearly, make neutral zone not to that end drift, the OH of the penetrating film of groove on must in the unit interval, passing through ^-With H by the following penetrating film of groove ⁺Number equate OH in the top electrophoresis pipe just ^-Formed electric current I _OH ^-With H in the electrophoresis pipe of bottom ⁺Formed electric current I _H ⁺Equate.

That is: I _OH ^-=I _H ⁺(1)

If supposition is S perpendicular to the interior sectional area of electrophoresis pipe arbitrarily, the OH in the alkaline buffer on last groove and electrophoresis pipe top ^-Migration rate is μ under the electric field force effect _OH ^-, its activity is a _OH ^-, the ion electricity price is Z _OH ^-, unit charge with electric weight be e; The H of same groove down and electrophoresis pipe bottom ⁺Migration rate is μ _H ⁺, activity is a _H ⁺, the ion electricity price is Z _H ⁺, unit charge with electric weight be e.

Then have: I _OH ^-=S μ _OH ^-A _OH ^-Z _OH ^-E (2)

I _H ⁺=S·μ _H ⁺·a _H ⁺Z _H ⁺·e (3)

Because Z _OH ^-=Z _H ⁺So, with (2), (3) formula substitution (1) formula, put in order:

(a _OH ^-)/(a _H ⁺) = (μ _H ⁺)/(μ _OH ^-) (4)

Again because ion migration rate μ in electric field _±Be directly proportional with electric-field intensity,

That is: μ _±=U _±(dV _±)/(dl _±) (5)

U _±Be mobility, promptly under uniform temperature and concentration, electric potential gradient equals at 1 o'clock, the migration rate of charged ion in solution, (dV _±)/(dl _±) be the electric potential gradient in the tiny area of charged ion place.

Get by (5) formula: μ _H ⁺=U _H ⁺(dV _H ⁺)/(dl _h ⁺) (6)

μ _OH ^-=U _OH ^-· (dV _OH ^-)/(dl _OH ^-) (7)

With (6), (7) formula substitution (4) formula,

Then have: (a _OH ^-)/(a _H ⁺)=(U _H ⁺(dV _H ⁺)/(dl _h ⁺))/(U _OH ^-(dV _OH ^-)/(dl _OH ^-)) (8)

If the electric-field intensity of hypothesis in electrophoresis pipe upper and lower part is equal,

That is: (dV _H ⁺)/(dl _h ⁺)=(dV _OH ^-)/(dl _OH ^-) (9)

Then (8) formula is (a _H ⁺)/(a _OH ^-)=(U _OH ^-)/(U _H ⁺) (10)

We know in the time of 25 ℃, (OH in the aqueous solution ^-) (H ⁺)=10 ^-14

More definite is: a _OH ^-A _H ⁺=10 ^-14(11)

Get by (10), (11) formula: a _H ⁺=(U _OH ^-)/(U _H ⁺) (10 ^-14)/(a ' _H ⁺) (12)

In (12) formula, a ' _H ⁺Be hydrionic activity in the last groove buffer solution.Getting (12) formula both sides with 10 is the negative logarithm at the end.

Put in order:

（－lga _H ⁺)=(14+lg (U _H ⁺)/(U _OH ^-) )-(－lga′ _H ⁺) （13）

That is: P ^H _Acid=(14+lg (U _H ⁺)/(U _OH ^-))-P ^H _Alkali(14)

Change in room temperature under the situation of little (about), can make constant K, make as 25 ℃:

K=14+lg (U _H ⁺)/(U _OH ^-) (15)

(as, in the time of 25 ℃, in extremely rare solution, U _H ⁺=3.630 * 10 ^-7Rice ²Second ^-1Volt ^-1, U _OH ^-=2.052 * 10 ^-7Rice ²Second ^-1Volt ^{-1 (1)}, K=14.255 then)

Then (14) formula becomes: PH _Acid=K-PH _Alkali(16)

(16) formula explanation is under the little situation of the temperature of buffer solution and ionic strength, in case the PH of buffer solution in the alkaline groove _AlkaliDecide, so the buffer solution ph in the acid tank _AcidAlso decide vice versa.

In keeping the neutral zone ballast, another important factor is the buffer capacity of buffer solution, and this is because in the aqueous solution in all charged ions.OH ^-And H ⁺The mobility maximum, this can obviously find out from table 1.

Table 1, when 298.15K, some ions are at the concentration of infinite dilution (rice ³Second ^-1Volt ^-1)

Cation	U o+Anion U o-
		H +K +B a 2+N a +L i +	36.30×10 -8?????OH -?????20.52×10 -87.62×10 -8??????SO 4 2-?????8.27×10 -86.59×10 -8??????Cl -?????7.91×10 -85.19×10 -8??????NO - 3?????7.40×10 -84.01×10 -8?????HCO 3 -?????4.61×10 -8

We know, when (16) formula of derivation, have ignored OH ^-And H ⁺Diffusion, ignore diffusion influence, just require OH ^-And H ⁺Concentration very low (because ions diffusion rate be directly proportional) with its concentration difference.The way it goes, because in preparation during buffer solution, we require total ionic strength is not too high, and the pH value of alkaline buffer is not more than 11, and the pH value of acidic buffer is not less than 3, and this explanation is the (H in the groove down ⁺)＜10 ^-3(OH in the M, last groove ^-)＜10 ^-3M.If we consider (OH ^-) and (H ⁺) to the influence of neutral zone.So just with (OH ^-) and (H ⁺) increase, neutral zone is corresponding to broaden, with (OH ^-) and (H ⁺) descend, neutral zone is corresponding to narrow down.

In addition, the amphiprotic substance that separate is also influential to neutral zone.Because they also have buffer capacity, particularly the amphiprotic substance that is separated is dense, under the very big situation of application of sample.

In order to ensure forming stable neutral zone, design in the equivalent again and detection technique, its method is that the amphiprotic substance in Fig. 6 is not a material to be separated, but very pure zwitterionic dyestuff, its isoelectric point (PI) is near neutral, (as Congo red PI 5.80 ⁽¹⁶⁾), the result of its electrophoresis of connection power supply:

Congo redly do not produce drift, groove H is described down ⁺And OH ^-The equivalent neutralization.

Congo red downward groove drift illustrates down groove H ⁺Concentration is big inadequately, and improving down, groove delays H in the buffer solution ⁺Concentration, or reduce to go up OH in the groove buffering ^-Concentration.

The drift of Congo red upwards groove, situation is just in time with above-mentioned opposite.

Should be noted that at H ⁺And OH ^-Can not wait number neutralization and when needing the concentration of the upper and lower groove buffer solution of more accurate adjustment, also will annotate adjustment than electricity.

In the process of (16) formula of derivation, suppose that the electric-field intensity on electrophoresis pipe bottom and top equates.

That is:

(dV _H ⁺)/(dl _H+) = (dV _OH ^-)/(dl _OH ^-) (9)

So, how to make (9) formula keep equating?

As everyone knows: V=1R (17)

That is: V=1/L (18)

So, total electrophoresis is I in the electrophoresis pipe if establish _Always, total electricity on electrophoresis pipe top is led and is L _{Total OH} ^-, total electricity of electrophoresis pipe bottom is led and is L _{Total H} ⁺

Then have: V _OH ^-=I _Always/ L _{Total OH} ^-(19)

V _H ⁺=I _Always/ L _{Total H} ⁺(20)

Again because: L=LS/1 (21)

So, be L if establish always leading in the electrophoresis pipe top (or going up groove) than electricity _{Total OH} ^-, highly be I _OH ^-; Electrophoresis pipe bottom (or following groove) is always led than electricity and is L _{Total H} ⁺Highly be I _H ⁺,

Then have: L _{Total OH} ^-=L _{Total OH} ^-S/I _OH ^-(22)

L _{Total H} ⁺=L _{Total H} ⁺S/I _H ⁺(23)

By: (9), (19), (20), (22), (23) formula are derived

: L _{Total OH} ^-=L _{Total H} ⁺(24)

If leading, the ratio electricity that forms is L after last groove (or electrophoresis pipe top) buffering is to reagent ionization _OH ^-, other non-bufferings form after to reagent ionization to lead than electricity and are L _OH ^-

Then approximate have: L _{Total OH} ^-=L _OH ^-+ L ' _OH ^-(25)

Also having in the groove (or electrophoresis pipe bottom) down equally:

L _{Total H} ⁺=L _H ⁺+ L ' _H ⁺(26)

By (24), (25), (26) formula,

L _OH ^-= L _H ⁺+( L′ _H ⁺- L′ _OH ^-) (27)

Order: △ L=L ' _H ⁺-L ' _OH ^-(28)

Then have: L _OH ^-=L _H ⁺+ △ L (29)

In (29) formula,

Work as L _OH ^-=L _H ⁺The time, must be △ L=0, illustrating does not need to add the electrolyte of a certain amount of neutrality (as KNO in either party buffer solution ₃Deng)

Work as L _OH ^-＞L _H ⁺The time, must be that △ L＞0 explanation needs to add in the oxytropism buffer solution electrolyte of a certain amount of neutrality, adjusts to L _{Total OH} ^-=L _{Total H} ⁺

Work as L _OH ^-＜L _H ⁺The time, must be that △ L＜0 explanation needs to add the certain amount of neutral electrolyte in the alcaliotropism buffer solution, adjust to L _{Total OH} ^-=L _{Total H} ⁺

Should be noted that with the pH value of solution equally, the ratio electricity of solution is led the influence that also is subjected to room temperature and concentration, and this just requires when the electricity of measuring buffer solution with conductivity gauge is led, and the temperature of the temperature of the buffer solution of electrophoresis pipe when measuring about equally in the time of will keeping electrophoresis.

From (16) and (29) formula derivation explanation, when the preparation buffer solution, must satisfy these two conditions of (16) and (19) formula, and this is easy to all accomplish in general laboratory and biological products factory.

Above-mentioned derivation (16) and (29) formula have been ignored the influence of amphiprotic substance to neutral zone, but in the actual electrophoresis process, amphiprotic substance is influential (aforementioned) to neutral zone.

For the amphoteric admixture matter that is among Fig. 6, when electrophoresis, will how to move?

Might as well establish the amphiprotic substance of required separation at this, have by its isoelectric point difference:

Alkaline species (P ^IOH ^-＞7): P ^IOH ^- ₁P ^IOH ^- ₂P ^IOH ^- _nP ^IOH ^- _N+1N is a natural number, and P from left to right ^IOH ^-Be cumulative.

Acid class (P ^IH ⁺＜7): P ^IH ⁺ ₁P ^IH ⁺ ₂P ^IH ⁺ _mP ^IH ⁺ _M+1M is a natural number, and P from left to right ^IH ⁺Be decrescence.

When electrophoresis first, the pH value in the electrophoresis pipe bottom is: P ^HH ⁺ _I+1The pH value on electrophoresis pipe top is: P ^HOH ^- _i, and the buffer solution in the electrophoresis pipe upper and lower part satisfied (16) and two conditions of (29) two formulas, if exist this moment:

During electrophoresis, concerning the amphiprotic substance of alkaline species, has only P so ^IOH ^- _N+1This group material just moves to negative electrode (being alkaline groove), and other material is stayed in the neutral zone, works as P ^IOH ^- _N+1When this group material moved to the penetrating film of alkaline groove (or go up groove), because the effect of penetrating film is just rested on here by obstruction, this group material separated with other amphiprotic substances like this.In acid class, has only P equally ^IH ⁺ _M+1This group material separates with other amphiprotic substances.Situation shown in Figure 8 will appear in the result of electrophoresis: divide from pH value, still have three district's bands, promptly alkaline band, neutral zone and acid band; And bring branch that P will be arranged from the district at amphiprotic substance place ^IOH ^- _N+1Band, alkaline blank tape, mixed zone, acid blank tape and P ^IH ⁺ _M+1Band.Employing certain way (as adopt 45 degree rotation modes in Fig. 1 and Fig. 3) can be with P ^IOH ^- _N+1And P ^IH ⁺ _M+1Band is separated.

Change the buffer solution of upper and lower groove.

Make:

Continue electrophoresis, P as a result ^IH ⁺ _mAnd P ^IOH ^- _nObtain equally separating.

Adopt and use the same method, can be with P ^IOH ^- _N-1, P ^IH ⁺ _M-1... P ^IOH ^- ₂, P ^IH ⁺ ₂P ^IOH ^- ₁P ^IH ⁺ ₁Separate, so just whole mixing amphiprotic substance be separated into 2(n+1)+a 1=2n+3 component or 2(m+1)+a 1=2m+3 component.

For separated amphiprotic substance such as protein, can measure its protein concentration with 751 spectrophotometers or protein nucleic acid detector or Kjeldahl, and measure its different biologically active as required.

It is to be noted P ^IH ⁺ _mAnd P ^IOH ^- _nThe amphiprotic substance that should regard as in certain pH value interval is being set up part jointly; When a certain pH value is constantly close in interval from its both sides in this pH value interval, just near the both sexes mixed composition of certain 1 PI; When the amphiprotic substance at this fixed point PI has only a kind of component, just become pure amphiprotic substance; When in this pH value interval during, represent no amphiprotic substance composition in the interval component of this pH value without any amphiprotic substance.

From above-mentioned situation as can be seen, the method of this separation amphiprotic substance resemble very much with a rod from two ends ceaselessly near the center with different cutting, so the method that amphiprotic substance is organized in this separation more calls two ends split plot design (The mathod of cutting at twoends) apart from difference.Clearly this method is applicable to separation, purifying and the analysis of not knowing the amphoteric admixture matter of isoelectric point to a group.

For a mixing amphiprotic substance, if, then can adopt PH clamping technology (The mathod of PH clamp) that this material is separated from this mixture in this isoelectric point of organizing known certain some component substance in mixture.For example known isoelectric point is P ^IH ⁺The amphiprotic substance of (being acid class) then when the buffer solution of preparation acid tank, at first is slightly less than P with pH value ^IH ⁺Buffer solution, simultaneously according to (16) or and (29) formula prepare the buffer solution of alkaline groove.PI＜the P as a result of electrophoresis ^IH ⁺Amphiprotic substance just can obtain separating.And then carry out the electrophoresis second time, at this moment the PH of the buffer solution of acid tank is larger than P ^IH ⁺, P behind the electrophoresis ^IH ⁺Just be separated mixed composition, be not difficult to find out, this method resembles with a pair of tongs P very much ^IH ⁺This component from mixed composition, clamp out.And this pliers PH just, so claim that this technology is PH clamping technology (The mathod of PH clamp).

Two kinds of methods that different isoelectric point amphoteric admixtures separate more than are described, in the split plot design of two ends,, just can estimate the isoelectric point of this amphiprotic substance if certain amphiprotic substance is segmented in a certain interval, if this pH value interval is enough little, just can record its PI.

Certainly, in two ends split plot design and PH clamping technology,, at this moment to make separated amphiprotic substance reach very pure degree if the amphiprotic substance that is separated might be impure, will combine with other isolation technics, this class technology has: ion-exchange, gel filtration, thin-layer chromatography etc.

But, with technical points such as ion-exchange, gel filtration from the Amphiphatic high polymer material, resemble protein, after wash-out, the OD at eluted protein peak often appears _ZBOBe worth very lowly, that is to say that protein concentration is low, also contain denseer salt composition in addition, these protein solutions are being carried out usually need to concentrate dialysis before the check and analysis.The method that concentrates is a lot, as the method that dries up of employing bag filter, and bag filter polyethylene glycol concentration method, the hyperfiltration method, method etc. is drained in vacuum refrigeration.

Utilize electrophoresis to concentrate at this, its principle is that the PH of preparation buffer solution departs from the PH of amphiprotic substance to be concentrated, make like this amphiprotic substance to be concentrated with net charge non-vanishing.Under effect of electric field, or to negative electrode, or anode moves, as shown in Figure 9, but because the logical effect of the resistance of penetrating film, and material to be concentrated is piled up at penetrating film place, remove out the solution that non-amphiprotic substance is piled up, both reached concentrated purpose, also reach the purpose of part dialysis.

Below in conjunction with a clinical examination example this method is described further.

In clinical examination, often need to measure the protein of hydrocrania, but because the protein concentration very low (20～40mg/dl) of hydrocrania ⁽¹⁸⁾So measuring needs to concentrate often ⁽¹⁹⁾Protein in hydrocrania, protein electrophoresis (filter paper method) ⁽¹⁸⁾Mainly be albumin globulin (α ₁, α ₂, β, γ), the PH of these protein is greater than 4, if be positioned over up and down and put into hydrocrania to be concentrated (wanting the centrifugal cell composition of removing earlier) in groove, the electrophoresis pipe so prepare the buffer solution of a pH value about 4, carry out electrophoresis, electrophoresis result, all protein will be assembled at the penetrating film of cathode terminal place, thereby reach concentrated purpose, adopt this method once can concentrate a plurality of samples.Similar application is urinated albumen in addition and is concentrated ⁽²⁰⁾Deng.

For the protein after concentrating with aforesaid way or other modes, before measuring its biologically active, need further with its protein solution in the electrolyte composition remove.The method that desalts is a lot, as dialysis, hyperfiltration method, gel filter method.At this electrodialysis method is discussed.

Referring to Figure 10, at last groove and following groove all is distilled water, is the protein that concentrates the back saliferous in the very short electrophoresis pipe in centre, behind the connection direct current electric source, micromolecular yin, yang ion passes penetrating film in effect of electric field, and macromolecular protein remains is stayed in the electrophoresis pipe.Electrophoresis is changed distilled water after a period of time, continues electrophoresis, all removes until the salt composition.Should be noted that the beginning electrophoresis when desalting, voltage can not be too high, so as not to produce very big electrophoresis (because of salt higher), can ceaselessly strengthen voltage later on.

Use

The method of multifunctional free-electrophoresis technology has given to describe in detail in principle, this using and wait in the electric weight and give explanation with the use of checkout gear the device of this electrophoretic techniques only.

The use of multifunctional free-electrophoresis technology device.

Referring to Fig. 1 and Fig. 3, during use, (J) pours down groove into towards liquid from concave surface at first to close down the acidity that the discharge opeing duct (e) of groove will prepare, and makes liquid level planoconcave (J), and notes draining down the gas of groove.Select suitable penetrating film (3) and put into concave surface (J), note exhaust, cover pressing plate (2), select the electrophoresis Guan Ji of suitable thickness as required, marking by conducting superposes one by one, form the electrophoresis pipe, groove buffer solution under each electrophoresis pipe bottom adds, and add a small amount of nearly neutral both sexes indicator therein.The electrophoresis Guan Ji of 45 degree rotation middle and upper parts, stagger in electrophoresis duct and bottom in the middle of making on the electrophoresis Guan Ji, then each the electrophoresis duct on the middle electrophoresis Guan Ji is added amphiprotic substance (sample) to be separated, 45 degree rotate top electrophoresis Guan Ji again, top electrophoresis and middle electrophoresis are staggered, fill it up with the alkaline buffer for preparing in electrophoresis duct, top to an electrophoresis pipe base concave surface (J) topmost, then selecting suitable penetrating film is positioned in the concave surface (J), cover pressing plate (attention exhaust), again by loading onto groove, in last groove, pour the buffer solution for preparing into, close the lid, will go up, the electrode cable of two grooves connects electrophoresis apparatus down, with exhaust regulator hole (n) on a flexible pipe one termination, the other end is clipped in pipe and goes up on the pipe clamp (g) of groove sidewall, and groove buffer solution under adding in flexible pipe makes the flat tank liquor face of going up of liquid level, middle and the top electrophoresis Guan Ji of rotation at last, reformulate a complete electrophoresis pipe, open the electrophoresis switch, carry out electrophoresis.

For the use of the cooling device among Fig. 3, the front is stated.

In the electrophoresis process, when the both sexes indicator is assembled near electrophoresis pipe central authorities, and stablize when motionless, show that electrophoresis finishes substantially, stop electrophoresis, 45 each adjacent electrophoresis Guan Ji of degree rotation stagger neighbouring electrophoresis duct mutually, can reach the separation purpose.

In the equivalent and the use of checkout gear:

With following groove buffer solution to be checked from following suitable for reading the pouring into of groove exhaust balance pipe, open piston, make down the groove buffer solution in observation tube, rest on certain altitude, closure piston, draw the both sexes indicator of a certain amount of nearly neutrality with the syringe that has lumbar puncture needle, to go up carefully in the groove exhaust balance pipe on the buffer solution liquid level that the both sexes indicator is added in the observation tube (amount can not be many), use the same method then to add and go up groove buffer solution to be checked, adjust respectively with buffer solution to be checked at last, liquid level in the following groove exhaust balance pipe is in same horizontal plane, open piston, connect direct power, observe the moving state of both sexes indicator in observation tube, judge whether equivalent neutralization (aforementioned).In the electrophoresis observation process, if the more cooling system of enabling of heat production.

Pluses and minuses relatively

The present invention has following advantage:

1, both can be used for the preparation of extensive amphiprotic substance, also can be used for separating and analysis of a small amount of and micro-amphiprotic substance.

2, institute's separate substance does not need to concentrate and just can reach very high concentration.

3, the cost of apparatus of the present invention is not high, and does not generally need valuable instrument and medicine when electrophoresis; In addition, the use of instrument is simple.

4, the material of Fen Liing is wide, both be adapted in other words molecular weight very high resemble DNA, RNA, even virus etc., also being adapted to the separation of materials such as very low polypeptide of molecular weight even amino acid, this has been avoided the limitation of the separate substance of a lot of electrophoretic techniques (resembling PAGE).

5, electrophoresis carries out in no support solution, and has rarer buffer solution to exist, so the time that electrophoresis is finished can be very not long.

6, the PH clamping technology is used to separate amphiprotic substance relative specificity, and clearly this technology is specially adapted to mass preparation.

7, before the separate substance, the preparation times such as preparation buffer solution generally need for a long time unlike ion-exchange and affinity chromatography etc.

8, convection action and electroosmosis to be separated and concentrated material result is influenced little because do not need very narrow district's band.

9, good concentrating function is arranged, this function is not only applicable to concentrating of amphiprotic substance, and can be used for clinical examination easily.

10, the total effects of electricity such as estimation and mensuration amphiprotic substance are arranged.

11, good electrodialysis effect is arranged.

12, the device of this multifunctional free-electrophoresis technology can be made different equipment series according to different needs (separating or a small amount of and micro-compartment analysis or concentrated dialysis as mass preparation).

Weak point is:

1, to variation of temperature reaction relatively large (this can overcome by cooling system)

2, to mutability inactivation under than strong basicity or acid situation, and its PI is at higher pH value or in the protein of low pH value, inapplicable present technique such as enzyme.

The specificity that 3, height can not be arranged as affinity chromatography.

4, can not carry out electrodialysis to uncharged little molecule.

Attached list of references:

1, volume such as Fu Xiancai: physical chemistry (volume two), 1980, Higher Education Publishing House.

2、Vesterberg，O.svensson，H.：Acta.chem，scand，20：820，1966

3、Svensson.H.：Acta.chem.Scclnd.15：325.1961

4, the biochemical microorganism of biology department of Zhongshan University teaching and research room compiles: biochemical technology introduction, 1979, People's Education Publishing House.

5、Bernfild.P.and??Nisselbanm.J.S.：J.Biol.chem.220：851，1956

6, volume such as Pan Jiaxiu: protein chemistry investigative technique, 1973, Science Press.

7、Depieds，Biffu.G.and??orsini.A.：compt.Rend.soc.Biol.152：1549，1958。

8, big gram is strong etc.: polyacrylamide gel electrophoresis, 1975, Science Press.

9、G.F.Bahr??ete：Micromethods??in??Molecular??Biology.vol.14，1973，Springer-verlag??Berlin.Heidelberg.New??York。

10, Zhang Longxiang etc.: biochemical test method and technology, 1982, People's Education Publishing House.

11、Bloemendal.H.J.Chromatography.2：121，1958

12、Reevs.R.E.etc：J.Amer.Soc.72：4773，1950

13、P.H.O′Farrell：J.Biol.chem.250：4007，1975

14、A.C.peacock??etc：Rioohem.7：688，1968

15、A.E.Bahlberg??etc：J.Mol.Biol.41：139，1969

16, the attached Long March of The 2nd Army Medical College institute: clinical immunology technology, 1980.

17, B.L. William Si etc.: practical biochemical theory and technology, 1979, Science Press.

18, Chen Guozhen chief editor: clinical practice, 1985, People's Health Publisher.

19, sieve pendant etc.: Chinese journal of medical examination, 12:303,1989.

20, Wang Yao etc.: Chinese journal of medical examination, 12:130,1989.

Claims (11)

1, a kind of multifunctional free-electrophoresis technology, it is characterized in that utilizing multifunctional free-electrophoresis device of the present invention to purify, and can concentrate and dialyse multiple amphiprotic substance to the separation that multiple amphoteric admixture carry out PH clamping technology (or two ends cutting techniques).

2, by the described multifunctional free-electrophoresis device of claim 1, it is characterized in that a section electrophoresis duct that is produced on regularly on the electrophoresis Guan Ji forms the electrophoresis pipe by stack, electrophoresis pipe two ends across penetrating film with have inert electrode on groove be connected with following groove.

3,, it is characterized in that superimposed electrophoresis Guan Ji can rotate again with protruding can accurately the coincideing of stable sliding mutually by circular stable sliding is recessed by the described multifunctional free-electrophoresis device of claim 2.

4,, it is characterized in that penetrating film does not have permeability to separating to purify or concentrate the amphiprotic substance of dialysing, and little molecule or small ion material are freely passed through by the described multifunctional free-electrophoresis device of claim 2.

5, by the described multifunctional free-electrophoresis device of claim 2, it is characterized in that whole electrophoretic apparatus can be designed to have the electrophoretic apparatus of cooling system.

6, by the described PH clamping technology of claim 1, it is characterized in that:

A, the used buffer solution of electrophoretic separation amphiprotic substance are pressed PH _Alkali=K-PH _AcidAnd L _OH=L _H+ △ L two formulas preparation two cover buffer solutions,

B, the formed PH scope of buffer solution of two inclined to one side alkali of PH or PH slant acidity comprises the isoelectric point that will isolate the amphiprotic substance component in two cover buffer solutions,

C, in use enables PH earlier away from neutral that cover buffer solution, after enable nearly that the neutral cover buffer solution of PH.

7, by the described two ends of claim 1 cutting techniques, it is characterized in that:

A, the used many covers buffer solution of electrophoretic separation amphiprotic substance are pressed PH _Alkali=K-PH _AcidAnd L _OH=L _HThe preparation of+△ L two formulas.

B, enable that neutral farthest cover buffer solution of PH when using these buffer solutions at first, that enables later the asymptotic neutrality of PH in proper order respectively overlaps buffer solution.

8, by the described multifunctional free-electrophoresis technology of claim 1, it is characterized in that in needs one equivalent and checkout gear, buffer solution is pressed PH _Alkali=K-PH _AcidAnd L _OH=L _HFurther checking of+△ L two formulas preparation.

9, press in the described equivalent of claim 8 and checkout gear.It is characterized in that graduated observation tube two ends respectively are communicated with a last groove and a following groove that has inert electrode, last groove respectively is connected an air guide balance pipe that internal diameter is identical again with following groove, and near observing the lower end piston is arranged.

10, press in claim 8, the 9 described equivalent and checkout gear, it is characterized in that around observation tube, can having cooling device.

11, by the described amphiprotic substance concentration method of claim 1, the pH value of used buffer solution when it is characterized in that electrophoresis departs from the isoelectric point of waiting to concentrate amphiprotic substance.

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