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Immobilized liposome capillary monolithic column preparation method and application

A capillary monolithic column and liposome technology, applied in the field of separation and analysis, can solve the problems of unfavorable compound separation, substance-biofilm interaction, insignificant difference in retention behavior, limited increase range, etc., and achieve good separation ability and long life , reduce the effect of adsorption

Active Publication Date: 2018-08-17
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the small specific surface area of ​​the inner wall of the capillary, the amount of liposomes immobilized on the inner wall of the capillary is very limited, resulting in a small ratio, small column capacity, and no significant difference in the retention behavior of substances on the liposome capillary open-tube column. , which is not conducive to applications such as compound separation, substance-biofilm interaction, etc.
We have tried to increase the specific surface area of ​​the inner wall of the capillary column by surface etching, and found that although the amount of immobilized liposomes has increased, the increase is very limited

Method used

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  • Immobilized liposome capillary monolithic column preparation method and application
  • Immobilized liposome capillary monolithic column preparation method and application
  • Immobilized liposome capillary monolithic column preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Preparation of liposomes.

[0031] 1. Prepare 4-hydroxyethylpiperazineethanesulfonic acid (HEPES) buffer

[0032] Accurately weigh 1.4894g of HEPES, add about 220mL of water to dissolve completely, adjust the pH to 7.40 with 1mol / L NaOH, add water to make up to 250mL, and obtain 25mmol / L HEPES buffer.

[0033] 2. Preparation of liposomes

[0034] (1) Accurately weigh 28.804 mg of phosphatidylcholine, dissolve it with 17 mL of chloroform:methanol solution (2:1, v / v), transfer it to a round-bottomed flask and evaporate under reduced pressure at 35°C until Form a uniformly dispersed film and dry it in vacuum for 12-16 hours to completely evaporate the residual organic solvent.

[0035] (2) Add about 7.6 mL of the above-prepared HEPES buffer solution into the flask, vortex to dissolve the film completely, and then form a multilamellar liposome solution. Using a cell ultrasonic breaker (200W; working time 3s; interval time 4s; ultrasonic frequency 99 times), the multi-cha...

Embodiment 2

[0038] Preparation of the immobilized liposome capillary monolithic column (the pressure applied to flush the capillary below is 50 psi).

[0039] 1. Take a new capillary and wash it with methanol for 30 minutes, 1 mol / L NaOH for 1 hour, 1 mol / L HCl for 30 minutes, and double distilled water until the effluent is neutral. 2 Dry under air at 120°C for 2 hours, and seal both ends with silicon rubber for later use.

[0040] 2. Weigh 50mg polyethylene glycol and 20mg urea, dissolve it with 500μL 0.01mol / L acetic acid solution, then slowly add 200μL tetramethoxysilane dropwise, stir vigorously in an ice-water bath, and inject it after the solution becomes a uniform sol state into the capillary; after reacting at 40°C for 12-16h, heat the capillary column at 120°C for 3h to expand the pores; then wash the capillary with methanol and water in sequence, 2 After drying at 50°C under atmosphere for 6h, it was raised from 50°C to 180°C at a rate of 2°C / min and kept for 1h, and finally h...

Embodiment 3

[0044] Performance testing of capillary monolithic columns of immobilized liposomes.

[0045] Electrochromatographic separation conditions: the total length of the immobilized liposome capillary monolithic column is 40.2cm, of which the effective length is 30cm; the electrophoresis buffer is 20mM phosphate buffer with pH 7.4; the sample injection condition is 3s under 5kV; the column temperature The temperature is 20°C, and the separation voltage is 20kV; when separating, add 50psi pressure at both ends of the capillary to prevent the generation of air bubbles. Thiourea neutral compound was used as a sample to determine electroosmotic flow (EOF).

[0046] Intra-day stability test: In the same immobilized liposome capillary monolithic column, the thiourea sample was repeatedly injected 30 times, and the relative standard deviation (RSD) of the measured EOF was 2.61% (n=30). The results showed that the prepared immobilized liposome capillary monolith column had good daily stabi...

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Abstract

The invention relates to an immobilized liposome capillary monolithic column preparation method and application thereof. On the surface of a silica gel matrix of a capillary monolithic column, liposome is immobilized by a physical coating method. The immobilized liposome capillary monolithic column preparation method disclosed by the invention has the advantages of good repeatability and large liposome immobilizing amount; the prepared monolithic column has the advantages of stable performance and longer service life. The immobilized liposome capillary monolithic column shows excellent separation performance on neutral compound of naphthol, benzene series, steroid medicine, carbamate and the like, can achieve base line separation on three proteins of lysozyme, trypsin, bovine serum albuminand obviously improves the adsorption phenomenon of the proteins on the capillary silica gel monolithic column; furthermore, retention factors of all the substances on the immobilized liposome capillary monolithic column are obviously enlarged, and retention behavior difference is more obvious. Therefore, an interaction strength effect between representation substances of the immobilized liposomecapillary monolithic column and a biological membrane is better.

Description

technical field [0001] The invention belongs to the technical field of separation and analysis, and in particular relates to a preparation method and application of an immobilized liposome capillary integral column. Background technique [0002] Capillary electrochromatography (CEC) is a micro-separation analysis technique developed in the 1990s. Due to its combination of the high efficiency of capillary electrophoresis and the high selectivity of liquid chromatography, it has attracted much attention in the field of separation analysis. The capillary electrochromatographic column is the most important part of the CEC system. According to the state of the stationary phase in the capillary, capillary electrochromatographic columns can be divided into capillary packed columns, capillary open-tube columns and capillary monolithic columns. The capillary packed column is obtained by filling the stationary phase into the capillary through professional plugging and packing proced...

Claims

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Application Information

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IPC IPC(8): B01J20/10B01J20/28B01J20/32B01D15/22B01D15/20B01D15/10C07K1/22
CPCB01D15/10B01D15/20B01D15/22B01J20/10B01J20/28014B01J20/3289C07K1/22
Inventor 肖玉秀王璇璇
Owner WUHAN UNIV
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