CN107449819B - A kind of anti-protein adsorption capillary column and preparation method thereof - Google Patents

Abstract

The invention relates to an anti-protein adsorption capillary column. The inner wall of the capillary is modified with several anti-protein adsorption covalent bonding coatings, and the anti-protein covalent bonding coatings are composed of photosensitive diazo resin and carboxylated chitosan The sugar is formed by photocuring and cross-linking reaction under ultraviolet light irradiation, and the thickness of each layer of the anti-protein covalent bonding coating is 1.1-1.8 nm. The preparation method uses carboxylated chitosan and photosensitive diazo resin to electrostatically self-assemble and photo-curing and cross-link to form a covalently bonded anti-protein adsorption coating. The separation efficiency of the prepared anti-protein adsorption capillary column is significantly improved, and the anti-protein adsorption performance is also relatively good. The preparation process of this method is relatively simple and easy to operate, low cost, good repeatability, non-toxic, safe and environmentally friendly, and has good stability and anti-protein adsorption performance, and can be used for the separation and detection of proteins, enzymes, peptide chains and hormones. purification.

Description

A kind of anti-protein adsorption capillary column and preparation method thereof

technical field

The invention relates to the technical field of capillary column separation, in particular to an anti-protein adsorption capillary column and a preparation method of the capillary column.

Background technique

In recent years, capillary electrophoresis has become more and more widely used in the analysis of proteins, biopolymers or drugs, and has become a modern and highly efficient separation method. However, with the development of this technology, there are problems that need to be overcome. There are also more and more, such as its poor repeatability, significant peak tailing, short service life, adsorption of the sample on the inner wall of the capillary, and so on. Some of the above problems can be effectively solved by changing the concentration and pH of the buffer, or modifying the capillary dynamic coating by non-covalent or covalent bonding.

However, non-covalently bonded coatings suffer from generally shorter lifetimes and lower separation efficiency, compared to covalently bonded coatings with longer lifetimes, better stability and higher separation efficiency. However, the preparation process of covalently bonded coatings is usually complicated, time-consuming and labor-intensive, and also involves complex reactions such as capillary silanization, and some of them are expensive or toxic, flammable and explosive. Hazardous reagents such as these bring great resistance to the capillary covalently bonded coating. For example: (1) Gerald Kleindiens et al reported in "Electrophoresis" 1998, 19, 262-269 that polystyrene (PS) derivatives and silanization reagents were used to prepare capillary electrophoresis-resistant covalently bonded coated columns for protein adsorption. The production process is more complicated, the required medicines are many, the cost is expensive, and the tetrahydrofuran used has toxicity; ) of the silylation reagent to prepare a capillary chip electrophoresis-resistant covalently-bonded coated column for protein adsorption. The preparation process takes a long time, and the silylation reagent used is toxic, easily hydrolyzed and expensive. (3) Xiaofang Fu et al. reported the use of carboxymethyl chitosan coating in Electrophoresis 2007, 28, 1958–1963. Although its preparation process is simple and easy to operate, the silylation reagent used contains acetonitrile (ACN). It is also a toxic and flammable reagent, and has the potential of explosion, which is dangerous for the conduct of experiments.

For the preparation methods of capillary electrophoresis covalently bonded coated columns listed above, some preparation processes are complicated and difficult to operate, some reagents are expensive, the cost is too high, the production process is toxic or flammable, and some coating quality It is unstable and the separation efficiency is not high. These problems have greatly limited the application of capillary electrophoresis in protein analysis.

SUMMARY OF THE INVENTION

Therefore, the technical problem to be solved by the present invention is that the existing preparation process is complicated, the operating conditions are harsh, the production process is toxic, the production efficiency is low, the production cost is high, and the prepared anti-protein adsorption capillary electrophoresis covalently bonded coating column The problem of poor quality is further provided, and an anti-protein adsorption capillary column and a preparation method thereof are further provided, the technological process is simple, the production process is environmentally friendly, the production efficiency is high, the production cost is low, and the prepared anti-protein adsorption capillary electrophoresis Covalently bonded coating The column is of good quality.

In order to solve the above-mentioned technical problems, the present invention adopts the following technical solutions:

An anti-protein adsorption capillary column, the inner wall of the capillary is modified with several anti-protein adsorption covalent bonding coatings, the anti-protein covalent bonding coating is made of photosensitive diazo resin and carboxylated chitosan through ultraviolet The photo-curing cross-linking reaction occurs after light irradiation, and the thickness of each layer of the anti-protein covalent bonding coating is 1.1-1.8 nm.

A preparation method of an anti-protein adsorption capillary column, comprising the following steps:

S1. A photosensitive diazo resin solution with a concentration of 0.05-500 mg·ml ^-1 , water, an aqueous solution of carboxylated chitosan with a concentration of 0.05-500 mg·ml ^-1 and water are slowly injected into the capillary; the photosensitive diazo resin and the mass ratio of the carboxylated chitosan aqueous solution is 1:1.0～1:1.5;

S2. Drying the capillary tube prepared in step S1, exposing it under an ultraviolet lamp with a wavelength of 193nm-400nm, and the exposure dose is 5-20000mJ/cm ² , to construct a diazo resin-carboxylated chitosan composition Covalently bonded coated anti-protein adsorption capillary column.

The weight-average molecular weight Mw of the photosensitive diazo resin is 700-5500, preferably 750-4000; the weight-average molecular weight Mw of the carboxylated chitosan is 100,000-250,000, preferably 150,000-200,000.

Preferably, the concentration of the photosensitive diazo resin solution is 0.1 mg/ml to 100 mg/ml, and the concentration of the carboxylated chitosan aqueous solution is 0.1 mg/ml to 100 mg/ml.

The step S1 is carried out at a temperature of 1 to 35°C, and the step S1 is repeated 1-5 times, preferably 1-3 times.

The step S2 is: drying the capillary prepared in the step S1, and exposing it under an ultraviolet lamp with a wavelength of 248 nm to 365 nm, and the exposure dose is 30-6000 mJ/cm ² , so that a diazo resin-carboxylation can be constructed. Anti-protein adsorption capillary column coated with chitosan covalently bonded.

The preparation method of the described anti-protein adsorption capillary column also comprises the following steps:

S0, internal activation pretreatment of capillary

Rinse the capillary with strong alkali solution, water, hydrochloric acid solution, water and methanol successively for 25-35min, 8-12min, 25-35min, 8-12min and 8-12min respectively, and then dry the capillary with inert gas, The activated capillary can be obtained;

The concentration of the sodium hydroxide solution is 0.09-0.11 mol·L ^-1 , and the concentration of the hydrochloric acid solution is 0.09-0.11 mol·L ^-1 .

The above-mentioned technical scheme of the present invention has the following advantages compared with the prior art:

(1) The inner wall of the anti-protein adsorption capillary column provided by the present invention is modified with an anti-protein adsorption covalent bonding coating, and the anti-protein covalent bonding coating is made of photosensitive diazo resin and carboxylated chitosan through UV-irradiated light-curing cross-linking reaction occurs, that is, photosensitive diazo resin and carboxylated chitosan photo-curing cross-linking reaction to form a covalent bond, which is not prone to quality problems such as capillary blockage, so it has excellent anti-protein adsorption performance.

(2) The preparation method of the anti-protein adsorption capillary column of the present invention utilizes photosensitive diazo resin and carboxylated chitosan to carry out electrostatic self-assembly to generate a photocuring cross-linking reaction to construct a stable anti-protein adsorption covalently bonded coating on the inner wall of the capillary . Specifically, the photosensitive diazo resin solution, water, aqueous solution of carboxylated chitosan and water were slowly injected into the capillary tube successively, and the photosensitive diazo resin solution and the carboxylated chitosan solution were successively followed by the driving force of the syringe. Assembled and grown along the inner wall of the quartz capillary, after ultraviolet light irradiation, the photosensitive diazo resin reacted with the silanols on the inner wall of the capillary and the sulfhydryls on the carboxylated chitosan to form a coating by photocuring and crosslinking. The preparation of the coating can be carried out in the aqueous phase by means of self-assembly, and the preparation process is simple and quick. The chemical bonding of the coating is realized by the photocuring cross-linking reaction of photosensitive diazo resin and carboxylated chitosan, and it is not easy to have quality problems such as capillary blockage.

(2) The preparation method of the anti-protein adsorption capillary column provided by the present invention also includes the pretreatment method of the inner wall of the capillary, respectively flushing the capillary with sodium hydroxide solution, distilled water, hydrochloric acid solution, water and methanol, and then drying the capillary with an inert gas , After pretreatment and activation, the inner wall of the capillary exposes many silyl hydroxyl groups (Si-OH), which form hydrogen bonds with the photosensitive diazo resin DR, and are converted into covalent bonds after UV exposure.

(4) The coating on the inner wall of the capillary of the present invention is formed of carboxylated chitosan by photocuring and cross-linking of a photosensitive diazo resin after being irradiated with ultraviolet light, avoiding the use of highly toxic and expensive coupling agent silylation reagents, simplifying The process of preparing a capillary anti-protein adsorption covalently bonded coating has the advantages of good repeatability, low price of raw materials, and green production process.

(5) The anti-protein adsorption capillary electrophoresis covalently bonded coating column prepared by the method of the present invention can be used for electrophoretic separation, detection and purification of biological samples such as proteins, hormones, peptide chains, enzymes and the like.

Description of drawings

Figure 1 is a comparative analysis diagram of the electrophoretic separation performance of four common proteins.

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. The present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the concept of the invention to those skilled in the art, and the invention will only be defined by the appended claims.

The invention provides an anti-protein adsorption capillary column, the inner wall of the capillary is modified with several anti-protein adsorption covalent bonding coatings, and the anti-protein covalent bonding coating is composed of photosensitive diazo resin and carboxylated shell The polysaccharide is formed by photo-curing and cross-linking reaction under ultraviolet light irradiation, and the thickness of each layer of the anti-protein covalent bonding coating is 1.1-1.8 nm.

The preparation method of the above-mentioned anti-protein adsorption capillary column uses diazo resin and carboxylated chitosan to self-assemble on the inner wall of the quartz capillary column layer by layer through electrostatic interaction, and undergoes a photo-curing cross-linking reaction through ultraviolet light, so that the original hydrogen The bonds are converted into covalent bonds, thereby forming a stable covalently bonded coating on the inner wall of the quartz capillary. Specifically, the described preparation method comprises the following steps:

S1. A photosensitive diazo resin solution with a concentration of 0.05-500 mg·ml ^-1 , water, an aqueous solution of carboxylated chitosan with a concentration of 0.05-500 mg·ml ^-1 and water are slowly injected into the capillary; the photosensitive diazo resin and the mass ratio of the carboxylated chitosan aqueous solution is 1:1.

The concentration of the above-mentioned diazo resin and carboxylated chitosan aqueous solution is 0.05-500mg/ml. If the concentration is too low, the self-assembly coating performance is not good. If the concentration is too high, the fluidity will be poor. The preferred concentration is 0.1 mg. /ml to 100mg/ml.

S2. Dry the capillary tube prepared in step S1, and expose it under an ultraviolet lamp with a wavelength of 193 nm to 365 nm, and the exposure dose is 5-20000 mJ/cm ² , and then a covalent diazo resin-carboxylated chitosan can be constructed. Bonded-coated anti-protein adsorption capillary column. If the wavelength of the exposure light source is too short or too long, and the exposure dose is too high or too low, the quality of the ^photocuring crosslinking of the coating will decrease. cm ² .

The weight-average molecular weight Mw of the photosensitive diazo resin is 700-5500. If the molecular weight is too low, the self-assembly coating performance will be poor. If the molecular weight is too high, the water solubility will be poor. The preferred molecular weight is 750-4000. The weight-average molecular weight Mw of the carboxylated chitosan is 100,000-250,000, and the preferred molecular weight is 150,000-200,000.

The step S1 is carried out at a temperature of 1 to 50°C. If the self-assembly temperature is too low, the aqueous solution is likely to freeze, and if the self-assembly temperature is too high, the diazo resin is easily pyrolyzed. The preferred self-assembly temperature is 1 to 35°C. °C.

The layer number of the layer-by-layer self-assembled composite structural coating of the diazo resin-carboxylated chitosan used in the present invention ranges from 2 to 12 layers, that is, the step S1 is repeated 1-5 times. If the number of layers is too small, the anti-protein adsorption performance will be poor, and if the number of layers is too large, the production efficiency will be reduced. Said, preferably repeated 1-3 times. If step S1 is repeated once, after UV exposure, two layers of anti-protein adsorption covalently bonded coatings are formed; if step S1 is repeated twice, after UV exposure, three layers of anti-protein adsorption covalently bonded coatings are formed, By analogy, step S1 is repeated 5 times, and 6 layers of anti-protein adsorption covalently bonded coatings are formed after UV exposure.

Described carboxylated chitosan has the structural formula shown in formula (1):

wherein n is an integer from 1007 to 1342.

Described photosensitive diazo resin has the structural formula shown in formula (2):

where n is an integer from 2 to 10.

The bare column of capillary column was washed with 0.09-0.11 mol·L ^-1 strong alkali solution, distilled water, 0.09-0.11 mol·L ^-1 hydrochloric acid solution, water and methanol, respectively, for 25-35min, 8-12min, 25-35min, 8-12min and 8-12min, and then dry the capillary with inert gas to get the activated capillary;

Preferably, the bare capillary column is washed with 0.1 mol·L ^-1 sodium hydroxide solution, distilled water, 0.1 mol·L ^-1 hydrochloric acid solution, distilled water and methanol successively for 30min, 10min, 30min, 10min and 10min respectively, Finally, dry the bare capillary column with N ₂ to obtain an activated bare capillary column.

The protein separation performance of the present invention is measured by CL-1020 type capillary electrophoresis instrument, and four proteins (1, lysozyme, 2, bovine serum albumin, 3, ribonuclease A, 4, myoglobin) in the analyte are The concentration of 0.5 mg/ml, the ultraviolet detection wavelength is 214 nm, the effective length of the anti-protein adsorption capillary column and the quartz capillary bare column in Example 3, that is, the total length of the capillary used in the separation experiment is 41 cm, and the inner diameter is 75 μm. , the column temperature was 25°C, the separation voltage was +15kV, and the separation medium was a phosphate buffer solution (pH=3.0) with a concentration of 40 mM.

Figure 1 shows the comparison results of protein separation between the bare capillary tube (curve A bare) and the coated capillary of the present invention (curve B covalent). As can be seen from the postal image, due to the strong adsorption between the protein and the inner wall of the capillary, there are only 2 characteristic peaks in the bare tube, and the peak shape is not good, and there is a tailing phenomenon, so a good separation cannot be achieved; Because it effectively inhibits the adsorption of proteins and the inner wall of the capillary, the four proteins can be completely separated within 20 minutes, and the stability is good.

Example 1

The present implementation provides a method for preparing an anti-protein adsorption capillary column, comprising the following steps:

S0, internal activation pretreatment of capillary

The capillary was washed successively with sodium hydroxide solution with a concentration of 0.1 mol·L ^-1 , distilled water, hydrochloric acid solution with a concentration of 0.1 mol·L ^-1 , distilled water and methanol, and the washing time was 30min, 10min, 30min, 10min and 10min respectively. , and then blow-dry the capillary with _N2 to get the activated capillary.

S1. Rinse the capillary column for 10 minutes with a photosensitive diazo resin solution (molecular weight 1000) with a concentration of 0.1 mg·ml ^-1 at a temperature of 25° C., and rinse the capillary column for 2 minutes with distilled water. Then, the capillary column was rinsed for 10 minutes with an aqueous solution of carboxylated chitosan (weight average molecular weight 150,000) with a concentration of 0.5 mg·ml ^-1 , and then the capillary column was rinsed with distilled water for 2 minutes, thus completing an electrostatic self-assembly cycle, repeat the above process 1 time;

S2. Dry the capillary tube prepared in step S1 with compressed air, expose it under a UV lamp with a wavelength of 248 nm, and expose the capillary tube at a dose of 50 mJ/cm ² . 2-layer covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 2

The present implementation provides a method for preparing an anti-protein adsorption capillary column, comprising the following steps:

S0, internal activation pretreatment of capillary

The capillary was washed successively with sodium hydroxide solution with a concentration of 0.09 mol·L ^-1 , distilled water, hydrochloric acid solution with a concentration of 0.11 mol·L ^-1 , distilled water and methanol, respectively, for 35min, 8min, 35min, 8min and 8min respectively. , and then blow-dry the capillary with _N2 to get the activated capillary.

S1. Under the condition of temperature of 25°C, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 5500) with a concentration of 100 mg·ml ^-1 for 10 minutes, and rinse the capillary column with distilled water for 2 minutes, and then rinse the capillary column with a concentration of 100 mg·ml ^-1 of carboxylated chitosan aqueous solution (weight-average molecular weight 200,000) was used to rinse the capillary column for 10 minutes, and then the capillary column was rinsed with distilled water for 2 minutes; thus, 1 electrostatic self-assembly cycle was completed, and the above process was repeated 5 Second-rate;

S2. The capillary prepared in step S1 is blown dry with compressed air, and exposed under a UV lamp with a wavelength of 365 nm, and the exposure dose is 5000 mJ/cm ² , so that 6 layers of diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 3

The structure of the anti-protein adsorption capillary column of the present embodiment is the same as that of the embodiment, and its preparation method includes the following steps:

S0, internal activation pretreatment of capillary

The capillary was washed successively with sodium hydroxide solution with a concentration of 0.11 mol·L ^-1 , distilled water, hydrochloric acid solution with a concentration of 0.11 mol·L ^-1 , distilled water and methanol, and the washing time was 25min, 12min, 25min, 12min and 8min respectively. , and then blow-dry the capillary with _N2 to get the activated capillary.

S1. Under the condition of temperature of 25°C, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 5000) with a concentration of 10 mg·ml ^-1 for 10 minutes, rinse the capillary column with distilled water for 2 minutes, and then use a concentration of The capillary column was rinsed for 10 minutes with a 2 mg·ml ^-1 aqueous solution of carboxylated chitosan (weight average molecular weight 160,000), and then the capillary column was rinsed with distilled water for 2 minutes; thus, 1 electrostatic self-assembly cycle was completed; repeat the above process 2 Second-rate.

S2. Dry the capillary tube prepared in step S1 with compressed air, and expose it under an ultraviolet lamp with a wavelength of 275 nm, and the exposure dose is 100 mJ/cm ² , and then three layers with diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 4

The structure of the anti-protein adsorption capillary column of the present embodiment is the same as that of the embodiment, and its preparation method includes the following steps:

S0, internal activation pretreatment of capillary

The capillary was rinsed successively with sodium hydroxide solution with a concentration of 0.1 mol·L ^-1 , distilled water, hydrochloric acid solution with a concentration of 0.1 mol·L ^-1 , distilled water and methanol for 28min, 9min, 32min, 11min and 10min respectively. , and then blow-dry the capillary with _N2 to get the activated capillary.

S1. Under the condition of temperature of 25℃, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 4000) with a concentration of 50 mg·ml ^-1 for 10 minutes, and rinse the capillary column with distilled water for 2 minutes, and then rinse the capillary column with a concentration of 40 mg·ml ^-1 of carboxylated chitosan aqueous solution (weight average molecular weight 180000) was used to rinse the capillary column for 10 minutes, and then the capillary column was rinsed with distilled water for 2 minutes; thus, 1 electrostatic self-assembly cycle was completed; repeat the above process 4 Second-rate.

S2. Dry the capillary tube prepared in step S1 with compressed air, and expose it under an ultraviolet lamp with a wavelength of 325 nm, and the exposure dose is 1000 mJ/cm ² , so that 5 layers of diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 5

The structure of the anti-protein adsorption capillary column of the present embodiment is the same as that of the embodiment, and its preparation method includes the following steps:

S0, internal activation pretreatment of capillary

The capillary was rinsed successively with potassium hydroxide solution with a concentration of 0.1 mol·L ^-1 , distilled water, hydrochloric acid solution with a concentration of 0.1 mol·L ^-1 , distilled water and methanol for 18min, 10min, 31min, 10min and 10min respectively. , and then blow-dry the capillary with _N2 to get the activated capillary.

S1. Under the condition of temperature of 1℃, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 3000) with a concentration of 500 mg·ml ^-1 for 10 minutes, and rinse the capillary column with distilled water for 2 minutes, and then rinse the capillary column with a concentration of The capillary column (weight-average molecular weight 100,000) was rinsed for 10 minutes with 500 mg·ml ^-1 of carboxylated chitosan aqueous solution, and then the capillary column was rinsed with distilled water for 2 minutes; thus, 1 electrostatic self-assembly cycle was completed; repeat the above process 2 Second-rate.

S2. The capillary prepared in step S1 is blown dry with compressed air, and then exposed under an ultraviolet lamp with a wavelength of 195 nm, and the exposure dose is 20000 mJ/cm ² , and then three layers with diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 6

The structure of the anti-protein adsorption capillary column of the present embodiment is the same as that of the embodiment, and the preparation method thereof is the same as that of the embodiment 5, wherein step S1 and step S2 are:

S1. Under the condition of temperature of 30℃, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 750) with a concentration of 0.05 mg·ml ^-1 for 10 minutes, and rinse the capillary column with distilled water for 2 minutes, and then use the concentration Rinse the capillary column with a carboxylated chitosan aqueous solution of 400 mg·ml ^-1 (weight average molecular weight 250,000) for 10 minutes, and then rinse the capillary column with distilled water for 2 minutes; this completes one electrostatic self-assembly cycle; repeat the above process 1 time.

S2. Dry the capillary prepared in step S1 with compressed air, and expose it to a UV lamp with a wavelength of 365 nm at an exposure dose of 5 mJ/cm ² , and then two layers with diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 7

The structure of the anti-protein adsorption capillary column of the present embodiment is the same as that of the embodiment, and the preparation method thereof is the same as that of the embodiment 5, wherein step S1 and step S2 are:

S1. Under the condition of temperature of 35℃, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 700) with a concentration of 400 mg·ml ^-1 for 10 minutes, and rinse the capillary column with distilled water for 2 minutes, and then rinse the capillary column with a concentration of The capillary column was rinsed with 0.05 mg·ml ^-1 of carboxylated chitosan aqueous solution (weight average molecular weight 120,000) for 10 minutes, and then with distilled water for 2 minutes; thus, one electrostatic self-assembly cycle was completed; repeat the above process 5 times.

S2. Dry the capillary tube prepared in step S1 with compressed air, and expose it under a UV lamp with a wavelength of 365 nm, and the exposure dose is 30 mJ/cm ² , so that 6 layers of diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

Example 8

The structure of the anti-protein adsorption capillary column of the present embodiment is the same as that of the embodiment, and the preparation method thereof is the same as that of the embodiment 5, wherein step S1 and step S2 are:

S1. Under the condition of temperature of 50°C, rinse the capillary column with a photosensitive diazo resin solution (weight average molecular weight 2000) with a concentration of 300 mg·ml ^-1 for 10 minutes, and rinse the capillary column with distilled water for 2 minutes, and then rinse the capillary column with a concentration of 200 mg·ml ^-1 of carboxylated chitosan aqueous solution (weight-average molecular weight 100,000) was used to rinse the capillary column for 10 minutes, and then the capillary column was rinsed with distilled water for 2 minutes; thus, 1 electrostatic self-assembly cycle was completed; repeat the above process 2 Second-rate.

S2. The capillary prepared in step S1 is blown dry with compressed air, and exposed under a UV lamp with a wavelength of 280 nm, and the exposure dose is 6000 mJ/cm ² , so that three layers with diazo resin-carboxylated chitosan can be constructed. Covalently bonded coated anti-protein adsorption capillary column. The thickness of each layer of the covalently bonded coating is 1.1-1.8 nm.

Since the silanols on the inner wall of the quartz capillary are shielded by the covalent cross-linking reaction between the coating and the coating, the covalently bonded coating column has good anti-protein adsorption performance.

The above-mentioned embodiments are merely examples for clear illustration, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

Claims (7)

1. A method for preparing an anti-protein adsorption capillary column, which is characterized by comprising the following steps:

s0, capillary tube internal activation pretreatment

Washing the capillary tube with strong alkali solution, water, hydrochloric acid solution, water and methanol for 25-35min, 8-12min, 25-35min, 8-12min and 8-12min, respectively, and blow-drying the capillary tube with inert gas to obtain activated capillary tube;

the strong alkali solution is sodium hydroxide solution with the concentration of 0.09-0.11 mol.L^-1The concentration of the hydrochloric acid solution is 0.09-0.11 mol.L^-1；

S1, concentration 0.05-500mg/ml^-1The photosensitive diazo resin solution of (1) water with a concentration of 0.05-500mg/ml^-1The carboxylated chitosan aqueous solution and the water are sequentially and slowly injected into the capillary; the mass ratio of the photosensitive diazo resin to the carboxylated chitosan aqueous solution is 1: 1.0-1: 1.5;

s2, drying the capillary tube prepared in the step S1, and exposing the capillary tube under an ultraviolet lamp with the wavelength of 248 nm-365 nm at the exposure dose of 30-6000mJ/cm²Thus, the anti-protein adsorption capillary column with the diazo resin-carboxylated chitosan covalent bonding coating can be constructed.

2. The method for preparing the protein adsorption capillary column as claimed in claim 1, wherein the weight average molecular weight Mw of the photosensitive diazo resin is 700-5500, and the weight average molecular weight Mw of the carboxylated chitosan is 100000-250000.

3. The method for preparing the protein adsorption resistant capillary column of claim 2, wherein the weight average molecular weight Mw of the photosensitive diazo resin is 750-4000, and the weight average molecular weight Mw of the carboxylated chitosan is 150000 to 200000.

4. The method of preparing an anti-protein adsorption capillary column according to claim 3, wherein the concentration of the photosensitive diazo resin solution is 0.1mg/ml to 100mg/ml, and the concentration of the carboxylated chitosan aqueous solution is 0.1mg/ml to 100 mg/ml.

5. The method for preparing an anti-protein adsorption capillary column according to claim 4, wherein the step S1 is performed at a temperature of 1 to 35 ℃.

6. The method for preparing an anti-protein adsorption capillary column according to claim 5, wherein the step S1 is repeated 1-5 times.

7. The method for preparing an anti-protein adsorption capillary column according to claim 6, wherein the step S1 is repeated 1-3 times.

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