CN1736538A - Method for enhancing dynamic absorption volume of ion exchange absorbent of transverse electric field - Google Patents
Method for enhancing dynamic absorption volume of ion exchange absorbent of transverse electric field Download PDFInfo
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- CN1736538A CN1736538A CN 200510014696 CN200510014696A CN1736538A CN 1736538 A CN1736538 A CN 1736538A CN 200510014696 CN200510014696 CN 200510014696 CN 200510014696 A CN200510014696 A CN 200510014696A CN 1736538 A CN1736538 A CN 1736538A
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Abstract
The invention discloses a method for improving the fluidizing adsorption capacity of the biomacromolecule such as protein on the ion exchange sorbent with an alternating electric field. Said preparative electrochromatogram separation device comprises a gel chamber and electrode chambers at double- side. The method for separation of biomacromolecule with this device is characterized in that: acetate, phosphonate, hydrogen carbonate , or trishydroxymethylaminomethane- hydrochloric acid buffer liquid is prepared as a mobile phase, the flow velocity of the mobile phase being 10- 200cm/h and the temperature being 2- 25Deg. C; iso- period alternating electric field with a voltage of 1- 600V and a current alternating period of 2- 200 seconds is stressed at the two sides of gel chamber, the flow velocity of electrode liquid being 20- 400cm/h and the temperature being 4- 25 Deg. C; and the specimen solution is sampled by means of meeting head and the sampling is stopped when the penetration approaches among 2- 80% of the sample concentration. Invented is characterized in that the separating power is strong, the handling capacity is large, the applicability extensive, and it has an extensive application prospect in separating and purifying the biomacromolecule such as protein and nucleic acid.
Description
Technical field
The present invention relates to a kind of method of utilizing action of alternative electric field to improve the dynamic adsorption capacity of protein and other on ion exchange absorbent, belong to biological product processing and separation technology field.
Background technology
At present, in biological downstream product process, chromatography has the irreplaceable status of other separation means.But the aperture of most of chromatography medias is all in nanometer scale, and the mass tranfer coefficient of protein in above-mentioned medium had a strong impact on the mass transfer of large biological molecule in chromatography media than low 2-3 the order of magnitude of small-molecule substance.Therefore, strengthen medium the inside mass transfer, to improve its separative efficiency of handling large biological molecule be one of chromatography technology problem anxious to be solved.
At present, the thinking that addresses the above problem mainly contains two kinds:
The first, reduce the particle diameter of medium.It mainly is conceived to reduce the chromatography media particle diameter, mass-transfer efficiency is improved in shortening mass transfer path.Owing to adopted the medium of small particle diameter, so the back pressure of chromatographic column increases in the operating process, and the mechanical strength of medium is had higher requirement.This method is mainly used in the chromatography experiment of analytical scale at present.
The second, the exploitation macroporous matrix.In the chromatography media preparation process, add certain pore-foaming agent, when generating the nanoscale diffusion hole, form a certain amount of micron-sized opening.Solution in opening to flow in diffusion hole then with the diffusion way transmission to streamed.Convective flow still needs very big external pressure in the hole, and macroporous matrix will be sacrificed certain adsorption capacity for the reinforcement of mass transport process.
Recently, along with the deep development of capillary electric chromatogram theory and application, people's notice also begins to change over to electrokinetic phenomenas such as utilizing electric osmose and accelerates on mass transport process and the raising separative efficiency.Therefore, in conventional chromatogram, introduce external electric field, utilize electrokinetic phenomena (electric osmose, electrophoresis) strengthen between media particle/particle hole inner transmission matter becomes the third thinking gradually.Electroosmosis comes from solid and liquid junction potential is poor, is the phenomenon that liquid moves with respect to powered surfaces in the electric field.Under External Electrical Field, contain electrolytical solution and be called EOF (EOF) with respect to the motion of charged static phase, adopting EOF to promote the capillary electric chromatogram that solution moves is to analyze the big focus of one in the chromatogram research at present.The generation of EOF is relevant with electric double layer, for 1: 1 type electrolyte aqueous solution, when its concentration is respectively 1, during 100mmol/L, the thickness of electric double layer is respectively 10,1nm.Therefore under common liquid chromatogram study condition, except that between media particle, produce the EOF, also may produce EOF in the hole of medium.Utilize EOF as motive force, when duct radius during much larger than electric double layer thickness (getting final product greater than 20 generally speaking), EOF speed and duct size are irrelevant, also do not have counter-pressure.Prior theory research shows that also in the pressure-driven chromatogram, with reducing of chromatographic media aperture, the contribution of convective mass transfer descends rapidly in the hole, and for EOF, needs only the aperture much larger than electric double layer thickness, and then EOF and aperture are irrelevant.Therefore, adopt electronic transmission to strengthen the approach that gets a good chance of that mass transfer will be the extensive high performance liquid chromatography of development.
In the existing preparation type electrochromatographic technique a kind of multi-channel Flow Electrophoresis equipment is arranged, this equipment by the Liu Zheng of Tsing-Hua University etc. propose (ion-exchange chromatography separation process under the alternating electric field, the chemical industry journal, 2001,52:311-315).Its nucleus equipment is a kind of electrophoresis tank of five chambers, intermediate cavity dress anionite DEAE-Sepharose FF, and its both sides are respectively the sample introduction chambers and go out the sample chamber, intermediate cavity and sample introduction and going out between the sample chamber separated by nylon leaching net.Two ends be the positive and negative electrode chamber, they and sample introduction, go out and separate with gel mould between the sample chamber, alternating electric field is applied on the platinum electrode in the electrode chambers.During experiment, bovine serum albumin(BSA) (BSA) sample liquid is pumped into Sample Room, the cushioning liquid that does not contain BSA is pumped into electrode chamber, return the electrode solution storage bottle, after outgasing, supply to recycle from the electrode solution that electrode chamber flows out.The BSA sample liquid is all passed dielectric layer and is entered out specimen chamber, flows out into the part automatic collector by the outlet that goes out specimen chamber.Absorption feeds cleaning fluid and eluent successively and carries out drip washing and wash-out after finishing, and after elution process finishes, feeds regenerated liquid bed is regenerated.This shows that this also is a kind of axial electric field electrochromatography, the filler chamber in the middle of flow phase and electric field all pass in the same way.The author utilizes this equipment to investigate alternating electric field to the dynamic absorption behavior of bovine serum albumin(BSA) on DEAE-Sepharose FF.Result of study shows, extra electric field between particle and the EOF that is produced in the medium holes can quicken transmittance process between protein and chromatograph packing material, significantly improve the dynamic adsorption capacity of chromatogram packed bed.But this device structure is complicated, and chamber is more.The chamber of middle filling material is very thin, if increase the increase that this chamber will cause two interelectrode distances.
Summary of the invention
The purpose of this invention is to provide a kind of method of utilizing action of alternative electric field to improve the dynamic adsorption capacity of protein and other on ion exchange absorbent, it is that transverse electric field is combined with chromatographic isolation, utilizes electrokinetic phenomena (electric osmose, electrophoresis) to strengthen between media particle/hole inner transmission matter and achieving the above object.
The present invention is realized by following technical proposals.A kind of action of alternative electric field that utilizes improves the method for protein and other in ion-exchange absorption attached middle school adsorption capacity, described preparation type transverse electric field electrochromatography separation equipment, wherein transverse electric field is meant that direction of an electric field is vertical with liquid flow direction, electrochromatography comprises that the electrode chamber of gel chamber and both sides constitutes, and gel chamber is sealed groove to be arranged on both sides groove that symmetry is arranged, the groove inner platform and middlely be the lucite body of rectangle hollow; Be provided with in the groove have molecular cut off greater than 1000 daltonian, can free lamina membranacea by conductive ion; Respectively there is an electrode chamber both sides outside gel chamber; The electrode chamber inwall is the inertia platinum electrode fixedly; Be filled with in electrode chamber and the gel chamber and comprise acetate, phosphate, bicarbonate or trishydroxymethylaminomethane-hydrochloric acid solution; Adopt the method for this device separates large biological molecule, it is characterized in that, comprise following process: 1.0-20mmol/L acetate, phosphate, bicarbonate or tris-HCI buffer that preparation contains 0-100mmol/L sodium chloride are to flow mutually and electrode solution, and flowing, it is preceding through 0.45 μ m milipore filter and ultrasonic processing to use mutually; Large biological molecule is prepared into the solution that concentration is 0.1-100mg/ml in being dissolved in and flowing mutually; After the electrode chamber of gel chamber and both sides balanced each other with flowing, the ion exchange absorbent that will have quaternary amine, sulfonic group, diethyl ethylenediamine base modification group was filled into the gel chamber of electric chromatographic column by the method for gravity filling; Flow rate of mobile phase is 10-200cm/h, and temperature is 2-25 ℃; Apply electrode voltage in the gel chamber both sides and be 1-600 volt, electric current alternating cycles and be 2-200 second etc. the cycle alternating electric field, the electrode solution flow velocity is 20-400cm/h, temperature is 4-25 ℃; After treating system stability, sample to be separated is with the mode sample introduction of meeting head on, and chromatographic column flows out liquid device detection after testing; After the exit sample penetration reaches sample introduction concentration 2-80%; Use the phase buffer solution flushing chromatographic column that flows instead to remove the not protein of absorption; Cut off the electricity supply, use linear gradient elution method elution chromatography post instead, elution volume is a 2-20 column volume; Above-mentioned absorption and wash-out can replace continuously and carry out.
Above-mentioned optimal conditions: preparation contains 2.0-15mmol/L acetate, phosphate, bicarbonate or the tris-HCI buffer of 5-50mmol/L sodium chloride for flowing mutually and electrode solution; Large biological molecule is prepared into the solution that concentration is 0-5-50mg/ml in being dissolved in and flowing mutually; After the electrode chamber of gel chamber and both sides balances each other with flowing, ion exchange absorbent is filled into the gel chamber of electric chromatographic column by the method for gravity filling; Flow rate of mobile phase is 20-70cm/h, and temperature is 4-15 ℃; Apply electrode voltage in the gel chamber both sides and be 5-300 volt, electric current alternating cycles and be 4-120 second etc. the cycle alternating electric field, the electrode solution flow velocity is 40-300cm/h, temperature is 5-15 ℃; After reaching sample introduction concentration 5-40%, the exit sample penetration stops sample introduction; The used elution volume of elution chromatography post is a 5-15 column volume.
Below the present invention is described in detail:
Key technology of the present invention has 8 points: the one, and the laterally introducing of alternating electric field in the chromatogram, adopted a kind of rectangle chromatographic column of three chambers, the filler chamber of middle filling material and the electrode chamber of both sides, isolate by the plane lamina membranacea between filler chamber and the electrode chamber, electrode is set in the electrode chamber, electrode links to each other with external power, has just produced transverse electric field in the filler chamber.Two of key technology is that lamina membranacea is the hydrophily lamina membranacea with certain molecule interception capacity, it mainly acts on is to stop large biological molecule to enter the electrode chamber of both sides and conductive ion can freely pass through, the thickness of lamina membranacea is relevant with its inner Joule heat, the thin more Joule heat of lamina membranacea is more little, satisfies the flow hydraulic pressure of phase and the requirement of mechanical strength own but lamina membranacea also needs certain thickness (or needing certain thickness gripper shoe).Three of key technology is to wait the application of cycle alternating electric field, solves large biological molecule in the accumulation of isolating the lamina membranacea place with the cycle of grade, and the while has also solved in the axial electric field electrochromatography problems that the lasting application owing to electric field causes; The size in cycle is again to influence the horizontal electrokinetic migration of protein and the key factor of mass transfer rate.Four of key technology is that buffer solution is conventional chromatogram buffer solution, and the ionic strength of buffer solution is 0.5-100mmol/L, and excessive ionic strength causes falling sharply of electronic transmission with compression double electric layer thickness.Five of key technology is in order to guarantee in the operating process the stable of the electrical conductivity of buffer solution and pH in the chromatographic column, and electrode solution is identical with the buffer solution composition.Six of key technology is that the temperature of buffer solution and electrode solution must have the ability of necessarily slowing down joule heating effect in filler chamber and the electrode chamber.Seven of key technology be the selection of buffer solution and electrode solution flow velocity except that should satisfying separative efficiency and filler demands for bearing capacity, also to satisfy the requirement of eliminating Joule heat in filler chamber and the electrode chamber.Eight of key technology is selections of voltage, and the size of voltage directly influences horizontal electronic transmission, mass transfer rate and filler chamber and the interior joule heating effect of electrode chamber of protein.
Since on electrode, apply one with filler chamber in the perpendicular electric field of liquid flow path direction, so in this form electrochromatography, chromatographic column axially, liquid phase is by pressure-driven; In the horizontal, solute is subjected to the influence of horizontal electronic transmission.Fig. 1 has provided the application orientation and the charged solute migration schematic diagram of alternating electric field.Electric chromatographic column laterally, three main mass transport processes are arranged: the molecular diffusion of EOF, protein electrophorese and protein under External Electrical Field.Because EOF and electrophoretic velocity all increase with the increase of electric-field intensity, so the local mass flux of adsorbing medium also will increase with the increase of electric-field intensity, so can effectively improve mass transport process by the size of regulating electric-field intensity.In addition, the EOF that produces with dielectric surface in medium holes also can be by reducing the long-pending absorption of proteins capacity that increases of stagnant area and the effective contact surface of increase.
Transverse electric field of the present invention increases the advantage that the method for adsorption capacity has: filler is conventional chromatograph packing material, low price; Because the electronic transmission of main employing improves adsorption capacity, the operating pressure of chromatogram does not require, and is lower to the bearing capacity requirement of equipment; This method is owing to adopted the rectangle chromatographic column structure of this three chambers, and the electrode chamber of filler chamber and both sides is isolated by hydrophilic film, so the electrode solution that the electrolytic gas that produces in the electrode chamber can stably be recycled is taken away, the Joule heat that produces in the filler chamber can be driven by axial compressive force again, flowing of cooling taken away mutually, thereby solved Joule heat and the electrolytic gas problem in the electrochromatography well; Electronic transmittance process depends on electric-field intensity, can reach the raising mass transport process by regulating electric-field intensity.Under the chromatographic run condition, the adsorbent of surface charging lotus can adopt this method, as Ion Exchange Medium, affine adsorbing medium etc.Added electric field situation not relatively, under lower electric-field intensity (＜20V/cm) adsorption capacity can improve 2～5 times, can satisfy multiple adsorbing separation requirement, be with a wide range of applications at aspects such as the separation of protein and other, purifying.
Description of drawings:
Fig. 1 has provided workflow schematic diagram of the present invention.。
Fig. 2 has provided that bovine serum albumin(BSA) has on DEAE-Sepharose FF, the dynamic adsorption curve of normalization during no electric field.Wherein electric chromatographic column is of a size of 1.5 * 0.5 * 1.2cm, sample-loading buffer is that 3.9mmol/L trishydroxymethylaminomethane-47mmol/L glycine (pH8.2) contains 5.0mmol/L sodium chloride, flow velocity 80cm/h, packing layer electric-field intensity is as shown in scheming to go up, the current cycle cycle is 20 seconds, and the bovine serum albumin(BSA) feed concentration is 2mg/mL.
The specific embodiment
Following example will give further instruction to the present invention.
Embodiment 1
The dynamic adsorption curve electrochromatography filler chamber of bovine serum albumin(BSA) on DEAE-Sepharose FF is of a size of (long * wide * dark) 1.5 * 0.5 * 1.2cm under the different electric-field intensity, and level pad is the 3.9mmol/L trishydroxymethylaminomethane-47mmol/L glycine buffer (pH8.2) that contains 5.0mmol/L sodium chloride.Eluent and regenerated liquid are respectively and contain 0.5 and the 3.9mmol/L trishydroxymethylaminomethane-47mmol/L glycine buffer (pH8.2) of 1.0mol/L sodium chloride.The concentration of BSA sample solution is 2.0mg/mL.In the experimentation, at 6 ℃, then cooled off by mixture of ice and water by mobile phase by the cooler cooling for electrode solution.
Adopt settling methods with the DEAE-Sepharose FF of the pre-equilibration filler chamber of packing into.Before last sample, filler chamber earlier with the level pad balance near UV (280nm) absorption value is stabilized in baseline.Then, add alternating electric field, and add pre-cooled BSA sample liquid with constant flow rate in the gel column both sides.The concentration of exit protein is detected by online UV, when outlet UV absorption value reaches 70% of sample introduction protein UV, stops to go up sample.Use level pad flushing chromatographic column instead to remove the not BSA of absorption, outlet UV absorption value can descend gradually and near baseline, at this moment stop the power supply supply.Then, use linear gradient elution method elution chromatography post instead, elution volume is 15 column volumes.At last, with the regenerated liquid filler of regenerating, the level pad balance sysmte is used in the regeneration back, prepares breakthrough experiment next time.Described in 2 explanations of other experiment conditions such as accompanying drawing, the result as shown in Figure 2, the dynamic adsorption capacity under the electric field significantly improves (3～5 times).
Embodiment 2
Utilize DEAE-Sepharose FF separation bovine serum albumin(BSA) and immunoglobulin G mixture electrochromatography filler chamber to be of a size of (long * wide * dark) 3.0 * 0.5 * 1.2cm, level pad is the 3.9mmol/L trishydroxymethylaminomethane-47mmol/L glycine buffer (pH8.2) that contains 5.0mmol/L sodium chloride; Eluent and regenerated liquid are respectively and contain 0.5 and the 3.9mmol/L trishydroxymethylaminomethane-47mmol/L glycine buffer (pH8.2) of 1.0mol/L sodium chloride.Total concentration is that the protein mixture of 2.0mg/mL is to be mixed by bovine serum albumin(BSA) and immunoglobulin G sample liquid that equal-volume, concentration are 2.0mg/L.In the experimentation, at 6 ℃, then cooled off by mixture of ice and water by mobile phase by the cooler cooling for electrode solution.
Adopt filler (DEAE-Sepharose FF) the pack centre chamber of settling methods with pre-equilibration.Electrode voltage is 75 volts, electric current period of change 20 seconds.Chromatographic column by the level pad balance after, opening power laterally applies alternating electric field chromatographic column.Then, with the pre-cooled protein mixture 32mL of speed adding of flow velocity 160cm/h, use level pad flushing chromatographic column instead to remove the not protein of absorption.Outlet UV absorption value can descend gradually and near baseline, at this moment stop the power supply supply.Switch to eluent then, with linear gradient elution, elution volume is 13.3 column volumes.The cut of collecting is analyzed by SDS-PAGE.Experimental result: under the overload situation, obtain electrophoretically pure bovine serum albumin(BSA) and immunoglobulin G product.
Claims (2)
1. a transverse electric field improves the method for ion exchange absorbent dynamic adsorption capacity, described preparation type transverse electric field electrochromatography separation equipment, wherein transverse electric field is meant that direction of an electric field is vertical with liquid flow direction, electrochromatography comprises that the electrode chamber of gel chamber and both sides constitutes, and gel chamber is sealed groove to be arranged on both sides groove that symmetry is arranged, the groove inner platform and middlely be the lucite body of rectangle hollow; Be provided with in the groove have molecular cut off greater than 1000 daltonian, can free lamina membranacea by conductive ion; Respectively there is an electrode chamber both sides outside gel chamber; The electrode chamber inwall is the inertia platinum electrode fixedly; Be filled with in electrode chamber and the gel chamber and comprise acetate, phosphate, bicarbonate or trishydroxymethylaminomethane-hydrochloric acid solution; Adopt the method for this device separates large biological molecule, it is characterized in that, comprise following process: 1.0-20mmol/L acetate, phosphate, bicarbonate or tris-HCI buffer that preparation contains 0-100mmol/L sodium chloride are to flow mutually and electrode solution, and flowing, it is preceding through 0.45 μ m milipore filter and ultrasonic processing to use mutually; Large biological molecule is prepared into the solution that concentration is 0.1-100mg/ml in being dissolved in and flowing mutually; After the electrode chamber of gel chamber and both sides balanced each other with flowing, the ion exchange absorbent that will have quaternary amine, sulfonic group or diethyl ethylenediamine base modification group was filled into the gel chamber of electric chromatographic column by the method for gravity filling; Flow rate of mobile phase is 10-200cm/h, and temperature is 2-25 ℃; Apply electrode voltage in the gel chamber both sides and be 1-600 volt, electric current alternating cycles and be 2-200 second etc. the cycle alternating electric field, the electrode solution flow velocity is 20-400cm/h, temperature is 4-25 ℃; After treating system stability, sample to be separated is with the mode sample introduction of meeting head on, and chromatographic column flows out liquid device detection after testing; After the exit sample penetration reaches sample introduction concentration 2-80%; Use the phase buffer solution flushing chromatographic column that flows instead to remove the not protein of absorption; Cut off the electricity supply, use linear gradient elution method elution chromatography post instead, elution volume is a 2-20 column volume; Absorption and elution process can replace continuously carries out.
2. chromatography separating method according to claim 1 is characterized in that: flow mutually and electrode solution is that the concentration that contains 5-50mmol/L sodium chloride is the 2.0-15mmol/L buffer solution; Large biological molecule is prepared into the solution that concentration is 0.5-50mg/ml in being dissolved in and flowing mutually; Flow rate of mobile phase is 20-70cm/h, and temperature is 4-15 ℃; Apply electrode voltage in the gel chamber both sides and be 5-300 volt, electric current alternating cycles and be 4-120 second etc. the cycle alternating electric field, the electrode solution flow velocity is 40-300cm/h, temperature is 5-15 ℃.After reaching sample introduction concentration 5-40%, the exit sample penetration stops sample introduction; The used elution volume of elution chromatography post is a 5-15 column volume.
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| CN103691318A (en) * | 2014-01-07 | 2014-04-02 | 延边大学 | Micro-scale substance separating method and capillary column transverse eletrochromatography separating device |
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| US5133844A (en) * | 1990-03-15 | 1992-07-28 | United States Department Of Energy | Method of electric field flow fractionation wherein the polarity of the electric field is periodically reversed |
| CN1040117C (en) * | 1993-07-16 | 1998-10-07 | 清华大学 | Chromatograph-type electrophoresis separation method |
| CN1040152C (en) * | 1993-07-16 | 1998-10-07 | 清华大学 | Chromatoelectrophoresis separating equipment |
| CN1209624C (en) * | 2001-04-02 | 2005-07-06 | 王少坤 | Chromatographic electric field separating method and device |
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| CN103691318A (en) * | 2014-01-07 | 2014-04-02 | 延边大学 | Micro-scale substance separating method and capillary column transverse eletrochromatography separating device |
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