CN104655769A - Method using liquid chromatography, bimolecular layer and stationary phase - Google Patents

Abstract

The invention relates to a method for improving the service life of separated biological substances by liquid chromatography and reducing the preparation time and requirements of samples of separated biological substances. The present invention adds a bimolecular layer stabilized by a polymer to the surface of the stationary phase by performing a series of chemical modifications on the surface of the stationary phase. The risk of the stationary phase being contaminated and adsorbed by macromolecular proteins with complex structures is eliminated.

Description

A method using liquid chromatography bilayer stationary phase

technical field

The invention belongs to the field of liquid chromatography, and mainly relates to the improvement of the liquid chromatography method, including improving the tolerance of the chromatographic column packing (stationary phase), reducing the requirement for sample pretreatment so as to save time and increase the detection speed , while also reducing the replacement frequency of the guard column in front of the chromatographic column.

Background technique

There are many examples of protein separations using liquid chromatography, especially reversed-phase liquid chromatography. However, due to the complex molecular structure and different compositions of proteins, generally successful separation requires a lot of early attempts: such as the selection of stationary phase, mobile phase selection, and optimization of mobile phase concentration ratio.

In addition, preparations for purification of the protein to be isolated are essential. Since proteins are macromolecules, they are likely to bind non-specifically to the stationary phase, resulting in "contamination" of the stationary phase, and even residual protein affecting separation. Generally, the chromatographic column will first pretreat the sample and remove some pollutants through the guard column. However, some proteins are components of interest to researchers and should not be removed. This method is undoubtedly contrary to the purpose of the research. Failure to carry out these protection measures may result in reduced service life of the guard column or chromatographic column.

Contents of the invention

The purpose of the present invention is to provide a stationary phase that can avoid the pollution caused by non-specific binding of proteins as far as possible, and reduce the requirements for sample pretreatment, and improve the service life of chromatographic columns and guard columns. Methods.

The present invention solves the above problems and includes the following methods:

- Preparation of stationary phase;

-React the stationary phase with the coupling agent to activate the surface of the stationary phase;

-Select the amphoteric substance (X) and its hydrophilic end modified counterpart (Y, the amphoteric substance after X modification

substance), Y can react with the surface of the activated stationary phase to fix;

-Select polar or non-polar substances to modify the surface of the stationary phase according to the needs (Z, similar to reverse

The non-polar C18 of phase chromatographic stationary phase), and draw the ratio of Y and Z according to need, Y and Z

Reacts with the activated stationary phase as a reactant.

- Add X again to form a bilayer.

- Optionally, depending on the need for stability, in the aqueous mixture of stationary phase particles that form a bilayer

Add methacrylate monomers to the mixture, and carry out polymerization reaction after mixing fully, so that the bilayer is more

Stablize.

-Fill the modified stationary phase into the chromatographic column;

-Derivation of the method of separation using a chromatographic column in a conventional manner, such as determination of parameters such as mobile phase, flow rate, etc.

- Optionally, using monomers, such as methacrylates, the polymeric bilayer is more stable.

As a preferred embodiment, the stationary phase in (1) can be selected from conventional liquid chromatography stationary phase particles such as silicon dioxide and titanium dioxide.

As a preferred embodiment, the amphoteric substance described in (2) is a molecule with one end being lipophilic and the other end being hydrophilic, and the substance forms a bilayer.

As a preferred embodiment, the amphoteric substance in (2) can be represented by the following formula:

Wherein A is a Va group element, such as P, As, Bi; Q is an oxygen group element, selected from: S, Se, Te, O; R ² , R ³ are independently a hydrocarbon group of 6-20 carbon atoms, R ¹ It is an alkylene group with a carbon number of 0-2, R ⁴ , R ⁵ , and R ⁶ are independent hydrocarbon groups or hydrogen atoms, with a carbon number of 0-3, and the charge of the nitrogen atom varies with the number of hydrocarbon groups connected to it.

Among them, preferably, A is phosphorus, Q is oxygen, R4 is a methyl group, ^R5 , ^R6 are hydrogen atoms, and ^R1 contains an alkylene group with a carbon number of 2;

As a preferred embodiment, the group connected to the nitrogen atom of (I) can be further modified, and the modified amphoteric substance (Y) can react with the silicon coupling agent.

As a preferred embodiment, silica particles are selected as the stationary phase, and a coupling agent is used to activate its surface so that it can react with other reagents. The reaction of silicon coupling agent to the surface of silicon dioxide is common knowledge, and the reaction conditions and the reactive groups that need to be provided by silicon coupling agent all belong to the prior art, and can be selected according to needs, for example, γ-aminopropyl three Ethoxysilane (APTS), at this time, the reactive group in Y can be a carboxyl group, and a group that needs to provide surface polarity, such as C18 in reversed-phase chromatography, can also choose a group containing a carboxyl group.

As a preferred embodiment, according to the polarity of the required stationary phase, such as reversed-phase chromatography, use a C18 stationary phase (due to the carbon chain length of Y itself, preferably using a fatty acid with a carbon number of 20-80), add the stationary phase surface For the polar reagent (Z), the amphoteric substance (Y) can be added in proportion at this time, and the ratio of Y to Z depends entirely on the polarity of the stationary phase required by the chromatographer, and there is no special limitation.

As a preferred embodiment, first activate the coupling agent on the silica surface according to the above method, and then add the required substances Y and Z to react to achieve basic coverage of the silica surface, and then add the unmodified substance X, since XY has the same structure, a bilayer is formed. Filter or centrifuge the silica, wash it slowly with clean water for 3-5 times before use.

As a more preferred embodiment, in order to further enhance the stability of the bilayer, add ethylene glycol dimethacrylate and methacrylate to the cleaned silica, and then add the initiator AIBN , under UV light irradiation for cross-linking polymerization. Since ethylene glycol dimethacrylate and methacrylate can diffuse into the hydrophobic region formed by the bimolecules, the polymer network formed by the polymers locks the bimolecular layer, making its structure more stable. In general, if no polymerization is carried out, adding other surfactants to the solution will lead to the collapse of the bilayer, making the silica surface modification ineffective. This can be confirmed from light scattering experiments. After adding surfactants, many small Peaks due to micelles, which do not appear after polymerization.

After the polymerized silica is washed and dried with centrifugal water, it can be filled into liquid chromatography as a stationary phase.

Beneficial effects of the present invention:

(1) The use of bilayers can effectively avoid the erosion and interference of proteins with complex structures on the stationary phase and guard column;

(2) The bimolecular layer provides stable and long-term protection to the stationary phase after polymerization, increasing the service life of its separated protein;

(3) The coverage ratio of the bilayer and the group that provides the separation effect of the stationary phase can be adjusted according to the needs, and the user can make adjustments according to the actual situation, which is very flexible;

(4) Embodiments of the present invention can also reduce the time and requirements for sample processing, because the bimolecular layer provided by the present invention can effectively avoid non-specific binding of proteins, because the material forming the bimolecular layer itself is the biological cell membrane. One, does not cause protein aggregation and non-specific binding.

Description of drawings

Figure 1: 1-stationary phase, silica particles; 2-bimolecular layer; 3-as the stationary phase group required for separation, it can be hydrophilic or lipophilic, and the type and coverage can be determined according to needs. 1 and 2, 3 are coupled and connected through a coupling agent, and the chemical bonds are not shown.

Figure 2: Bilayers locked and stabilized by polymers

Detailed ways

Select silica particles as the stationary phase, wash with anhydrous solvent, and put it into an organic solvent after drying. The organic solvent is preferably free of water (toluene can be used), and it can react with the modified amphoteric substance (Y) by adding Silicon coupling agent (ATPS):

Amino groups are formed on the surface of the silica.

After the reaction is completed, centrifuge to separate the activated silica on the surface, and react with Y and fatty acid after drying (the fatty acid used is a straight-chain carboxylic acid containing 42 carbon atoms), and react in an organic solvent under heating conditions to form an amide bond Modify the amphoteric substances and non-polar groups on the surface of silica, centrifuge, wash and dry, mix with the unmodified amphoteric substance (X) in an organic solvent, the solvent is preferably chloroform, after uniform mixing, use an inert gas Dry as vibes.

Said Y is 16:0 Glutaryl PE:

1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(glutaryl)(sodium salt) 870245, from AVANTI Polar Lipids;

R4 of said X is a methyl group, R5 and R6 are hydrogen atoms, and other structures of X are the same as Y.

Add deionized water to the dried mixture and place in a sealed container.

Prepare a water bath with a temperature of 40-60 degrees Celsius, prepare a mixture of dry ice and isopropanol as an ice bath, and cool it to room temperature after several rounds of alternating temperature changes in the water bath and ice bath, centrifuge the silica solid, and use Wash the solid silica slowly with water for 3-5 times. At this time, a silica stationary phase with a bimolecular layer on the surface was obtained. Add ethylene glycol dimethacrylate and methacrylate to the above solution and mix well.

Add AIBN initiator to the above mixture, mix thoroughly for 30 minutes, react under ultraviolet irradiation for 30 minutes, then separate and wash by filtration, centrifugation, etc. to obtain stationary phase silica. According to the P and N content obtained by XPS surface analysis, the solid-phase silica has been successfully modified into a bimolecular layer surface, and after cleaning with a large amount of surfactant, no P and N elements were detected in the solution, indicating that the bimolecular The layer is very stable, because the general bilayer is formed and will be destroyed by surfactants to become micelles.

Therefore, the present invention makes the surface of the traditional silica stationary phase form a bimolecular layer that can resist the non-specific binding of proteins through a series of chemical modifications, which will reduce the requirement for sample pretreatment and shorten the time for liquid chromatography to separate biological substances. The overall time of the analysis is improved, and the lifetime of the stationary phase is extended.

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.