CN104655769A - Method using liquid chromatography, bimolecular layer and stationary phase - Google Patents
Method using liquid chromatography, bimolecular layer and stationary phase Download PDFInfo
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- CN104655769A CN104655769A CN201410797990.6A CN201410797990A CN104655769A CN 104655769 A CN104655769 A CN 104655769A CN 201410797990 A CN201410797990 A CN 201410797990A CN 104655769 A CN104655769 A CN 104655769A
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Abstract
The invention relates to a method using liquid chromatography to prolong the service life of a separated biological substance and to reduce and lower the preparation time and requirements for a separated biological substance sample. According to the method, a series of chemical modifications are performed on the surface of a stationary phase, a bimolecular layer stabilized by a polymer is added to the surface of the stationary phase, and the bimolecular layer cannot generate nonspecific binding with biomass such as a protein, so that the risk that the stationary phase is polluted and absorbed by a complicated macromolecule protein is reduced greatly.
Description
Technical field
The invention belongs to liquid chromatography field; relate generally to the improvement on liquid-phase chromatography method; comprise the tolerance improving chromatographic column filler (Stationary liquid); reduce and to reach to save time, the object of detection speed is speeded to the requirement of sample pretreatment, also reduce the replacement frequency of the guard column before to chromatographic column simultaneously.
Background technology
Use liquid chromatography isolated protein, especially reversed-phase liquid chromatography, has had many examples.But due to protein molecular structure complexity, forming different, general successful separation all needs carry out a large amount of trials early stage: such as to the selection of Stationary liquid, the selection of mobile phase, be optimized mobile phase concentration proportioning.
In addition, treat the preliminary work that isolated protein carries out purifying to be absolutely necessary.Because protein belongs to large molecule, probably with Stationary liquid generation non-specific binding, cause Stationary liquid to be subject to " pollution ", even cause the protein influence remained to be separated.First general chromatographic column all can carry out pre-service to sample, and removes part pollutant by guard column, but some protein is researcher, and interested component should not be removed, and this method is runed counter to research purpose undoubtedly.If do not carry out these protection work, then may cause reduce the serviceable life of guard column or chromatographic column.
Summary of the invention
No matter the object of the present invention is to provide a kind of is that polarity or non-polar stationary phase can avoid protein non-specific to combine the pollution brought as far as possible; and reduce requirement to sample pretreatment, and improve the method in serviceable life of chromatographic column, guard column.
The present invention solves the problem and comprises following methods:
-prepare Stationary liquid;
-Stationary liquid and coupling agent are reacted, activation Stationary liquid surface;
-select amphiprotic substance (X) and the adorned counterpart of water-wet side (Y, the both sexes after modifying by X
Material), Y can fix with the Stationary liquid surface reaction of activation;
-select to the Stationary liquid surface polarity of modifying or apolar substance that (Z is similar to anti-as required
The nonpolar C18 of phase chromatographic stationary phases), and draw the ratio of Y and Z as required, by Y and Z
Stationary liquid as reactant and activation reacts.
-add X again, form bilayer.
-can optionally according to the needs of stability, defining the aqueous mixtures of stationary-phase particle size of bilayer
In add the monomer of methyl acrylic ester, carry out polyreaction after mixing fully, be bilayer more
Stable.
-Stationary liquid after modifying is filled in chromatographic column;
-draw the method using chromatographic column to be separated in a conventional manner, such as determine the parameter such as mobile phase, flow velocity.
-optional, use monomer, such as methacrylate, gathering bilayer is more stable.
As preferred embodiment, in (1), Stationary liquid can select the conventional liquid phase such as silicon dioxide, titania chromatographic stationary phases particle.
As preferred embodiment, amphiprotic substance described in (2) is one end oleophylic, the molecule that the other end is hydrophilic, and this material forms bilayer.
As preferred embodiment, the amphiprotic substance in (2) can be expressed from the next:
Wherein A is Va elements, such as P, As, Bi; Q is oxygen group elements, is selected from: S, Se, Te, O; R
2, R
3be the alkyl of 6-20 carbon atom independently of each other, R
1for containing carbon number be the alkylidene of 0-2, R
4, R
5, R
6being respective independently alkyl or hydrogen atom, is 0-3 containing carbon number, and nitrogen-atoms electric charge connects alkyl quantity with it and changes.
Wherein preferred, A is phosphorus, and Q is oxygen, and R4 is methyl, R
5, R
6for hydrogen atom, R
1be the alkylidene of 2 containing carbon number;
As preferred embodiment, the group be connected with nitrogen-atoms of (I) can be modified further, and the amphiprotic substance (Y) after modification can react with silicone couplet.
As preferred embodiment, select silica dioxide granule as Stationary liquid, use coupling agent activate its surface, make its can with other reagent reactings.The reaction of silicone couplet to silica surface is common practise, reaction conditions and the reactive group needing silicone couplet to provide belong to prior art all, can select as required, such as can use gamma-aminopropyl-triethoxy-silane (APTS), reactive group now in Y can be carboxyl, and need the group that surface polarity is provided, the C18 of such as reverse-phase chromatography, also can select the group containing carboxyl.
As preferred embodiment, according to the polarity of required Stationary liquid, such as reverse-phase chromatography, use C18 Stationary liquid (due to the carbon chain lengths of Y itself, the fatty acid that preferred use is 20-80 containing carbon number), add Stationary liquid surface polarity reagent (Z), now proportionally can add amphiprotic substance (Y), the ratio of Y and Z depends on the Stationary liquid polarity required for personnel using chromatogram completely, has no particular limits.
As preferred embodiment, first silica surface is carried out coupling agent activation according to described method above, then add desired substance Y and Z and react, reach the basic covering to silica surface, add not modified material X subsequently, because XY structure is identical, form bilayer.By silica filtration or centrifuging, stand-by after slowly cleaning 3-5 time with clear water.
As preferred embodiment, in order to strengthen the stability of bilayer further, in the silicon dioxide after cleaning, add Ethylene glycol dimethacrylate and methacrylate, then when stirring, add initiator A IBN, under UV-irradiation, carry out cross-linked polymeric.What can be spread to that bimolecular becomes due to Ethylene glycol dimethacrylate and methacrylate detests pool, and bilayer is locked by the polymer network that therefore polymkeric substance is formed, and makes its structure become more stable.Generally, if be not polymerized, in solution, add other surfactants will cause bilayer to be disintegrated, silica surface is modified lost efficacy, this can be confirmed from light scattering experiments, there is the peak that many little micella micelle cause after adding surfactant, and there will not be this situation after polymerization.
Silicon dioxide after polymerization, after centrifugal clear water washing drying, can be inserted in liquid chromatography as Stationary liquid.
Beneficial effect of the present invention:
(1) bilayer is used can effectively to avoid the protein of labyrinth to the erosion of Stationary liquid, guard column and interference;
(2) this bilayer provides stable, the permanent protection to Stationary liquid after polymerization, increases the serviceable life of its isolated protein;
(3) coating ratio of bilayer can adjust completely as required with the group providing Stationary liquid to be separated effect, and user can need according to actual conditions and adjust, very flexibly;
(4) embodiments of the present invention can also reduce time to sample process and requirement, because bilayer provided by the invention effectively can avoid the non-specific binding of protein, because the material forming this bilayer inherently forms the one of biological cell membrane, protein can not be caused and build up and non-specific binding.
Accompanying drawing explanation
Accompanying drawing Fig. 1: 1-Stationary liquid, silica dioxide granule; 2-bilayer; 3-, as the Stationary liquid group needed for separation, can be hydrophilic or oleophylic, determine kind and coverage rate as required.Be connected by coupling agent generation coupling between 1 and 2,3, its chemical bond does not show.
Accompanying drawing Fig. 2: by the stable fixed bilayer of polymeric lock
Embodiment
Select silica dioxide granule as Stationary liquid, anhydrous solvent washs, and after drying, puts it in organic solvent, the preferred moisture-free of organic solvent (can toluene be used), add can with modify after the silicone couplet (ATPS) that reacts of amphiprotic substance (Y):
Silica surface forms amino.
After completion of the reaction, centrifuging goes out the silicon dioxide of surface through overactivation, with Y and fatty acid response (fatty acid used is the straight-chain carboxylic acid containing 42 carbon atoms) after dry, react in organic solvent in a heated condition, form amido link and amphiprotic substance and non-polar group are modified silica surface, after centrifugal clean dry, mix in organic solvent with not modified amphiprotic substance (X), the preferred chloroform of this solvent, after Homogeneous phase mixing, use inert gas dry as atmosphere.
Described Y is 16:0Glutaryl PE:
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl) (sodium salt) 870245, comes from AVANTI Polar Lipids;
The R4 of described X is methyl, and R5, R6 are hydrogen atom, and other structures of X are identical with Y.
In dried potpourri, add deionized water, be placed in the container of sealing.
Get out water-bath, temperature is at 40-60 degree Celsius, and the potpourri getting out dry ice and isopropyl alcohol, as ice bath, is cooled to room temperature after carrying out many wheels and replace alternating temperature in water-bath ice bath, centrifuging silica solid, slowly washs solid silica 3-5 time with clear water.Now obtain the silicon dioxide Stationary liquid that surface has bilayer.Add Ethylene glycol dimethacrylate and methacrylate to above-mentioned solution, fully mix.
In said mixture, add AIBN initiating agent, fully mixing 30 minutes, reacts 30 minutes under Ultraviolet radiation, then adopts filtration, the mode such as centrifugal is separated clean, be fixed aerosil.By the P that XPS surface analysis obtains, N content is known, solid phase silicon dioxide is successfully modified to bilayer surface, and after the cleaning of exhibiting high surface activating agent, P is not detected in solution, N element, illustrates that this bilayer is highly stable, this is because general bilayer can be destroyed by surfactant after being formed become micella micelle.
Therefore, the present invention makes traditional silicon dioxide Stationary liquid surface define the bilayer can resisted protein non-specific and combine by a series of chemical modification, this will make the requirement reduced during liquid chromatography separating bio material sample pretreatment, shorten the overall time analyzed, and extend the serviceable life of Stationary liquid.
Claims (7)
1.-mono-kind uses the method for liquid chromatography, it is characterized in that comprising the following steps:
-use coupling agent activation on its Stationary liquid surface;
-select to be formed the amphiprotic substance X of bilayer, its water-wet side modification made water-wet side can form chemical bond with the Stationary liquid surface reaction after coupling agent activates, modified amphiprotic substance is Y;
-select according to the polarity of wanted separate substance the Stationary liquid coating material Z determining Stationary liquid surface nature, this dressing agent contains the group that the Stationary liquid surface that can activate with coupling agent forms chemical bond;
-Y, Z are mixed in proportion react with Stationary liquid as required, make Y, Z covers Stationary liquid surface;
-add amphiprotic substance X, make X, Y shape becomes bilayer;
-add polymerisable monomer to the Stationary liquid aqueous dispersions containing bilayer, fully after diffusion mixing, carry out polyreaction, after being separated stationary-phase particle size, obtain the constitutionally stable Stationary liquid containing bilayer.
2.-method according to claim 1, is characterized in that Stationary liquid is silicon dioxide, coupling agent Wei γ ?aminopropyl triethoxysilane.
3. ?method according to claim 1, amphiprotic substance X be (I) formula definition compound:
Wherein A is the one of P, As, Bi; Q is oxygen group elements, is selected from: S, Se, Te, O; R
2, R
3be independently of each other 6 ?the alkyl of 20 carbon atoms, R
1for carbon number 0 ?2 alkylidene, R
4, R
5, R
6being respective independently alkyl or hydrogen atom, is 0 ?3 containing carbon number, and nitrogen-atoms electric charge connects alkyl quantity with it and changes.
4. ?method according to claim 3, wherein A is phosphorus, and Q is oxygen, R
4for methyl, R
5, R
6for hydrogen atom, R
1be the alkylidene of 2 containing carbon number.
5. ?method according to claim 1, wherein material Y is
The R of amphiprotic substance X
4for methyl, R
5, R
6be hydrogen atom, other structures are identical with Y.
6. ?method according to claim 1, described polymerisable monomer is the potpourri of Ethylene glycol dimethacrylate and methacrylate, and initiators for polymerization is AIBN.
7. ?method according to claim 1, Stationary liquid coating material Z be containing carbon number be the straight chain fatty acid of 20-80.
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| US20030159933A1 (en) * | 2000-06-03 | 2003-08-28 | Thomas Bayerl | Method for electrophoretically separating membrane proteins |
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