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Ion exchange chromatography fixed phase, preparing method and application of the same

An ion-exchange chromatography and stationary phase technology, applied in the field of ion-exchange chromatography stationary phase and its preparation and application, can solve the problem of low protein recovery rate and achieve improved separation selectivity, good biocompatibility and biological affinity, high The effect of mechanical strength

Inactive Publication Date: 2008-09-03
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, irreversible adsorption between proteins and polyethylene occurs due to hydrophobic interactions, resulting in poor protein recovery

Method used

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  • Ion exchange chromatography fixed phase, preparing method and application of the same
  • Ion exchange chromatography fixed phase, preparing method and application of the same

Examples

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Comparison scheme
Effect test

Embodiment 1

[0017] Example 1: Preparation of hydroxyapatite-coated zirconium-magnesium composite oxide ion-exchange chromatography stationary phase and separation of proteins

[0018] (1) Preparation of supersaturated calcium-phosphorus solution: Dissolve analytically pure sodium chloride, potassium chloride, disodium hydrogen phosphate, hydrochloric acid, and calcium chloride in water successively, and its ion concentrations are respectively [Na + ]=136.8mM, [K + ]=3.71mM, [Cl - ]=144.5mM, [Ca 2+ ] = 3.10 mM, [HPO 4 2- ] = 1.86 mM. The prepared solution was adjusted to pH 7.4 with trishydroxymethylaminomethane and stored at 5°C.

[0019] (2) Deposition and growth of hydroxyapatite coating: the zirconium-magnesium composite oxide was treated with 1M sodium hydroxide, washed until neutral, and dried at 160° C. for 8 hours. Next, immerse in 0.5M disodium hydrogen phosphate solution and saturated calcium hydroxide solution in turn, wash and dry at 120°C. Then soak it in 500mL supersat...

Embodiment 2

[0021] Example 2: Preparation of hydroxyapatite-coated silica gel microspheres ion-exchange chromatography stationary phase and separation of proteins

[0022] The 5 μm silica gel microspheres were treated with 1M hydrochloric acid, washed until neutral, and dried at 160°C for 8 hours. Soak it in 500mL of supersaturated calcium-phosphorus solution, keep the temperature at 36.5±0.5°C, take it out after soaking for a period of time, rinse it with secondary water, and dry it at 120°C. The above-mentioned materials were packed into a 150mm × 4.6mmi.d. chromatographic column, and with phosphate buffer solution as mobile phase, five kinds of proteins were separated in gradient mode (such as figure 2 ).

Embodiment 3

[0023] Example 3: Preparation of hydroxyapatite-coated capillary

[0024] Take the capillary, rinse with 1M sodium hydroxide solution, secondary water, 1M hydrochloric acid, and secondary water for 1 hour in sequence, and then dry at 120° C. for 4 hours under nitrogen. Under the action of gravity, the supersaturated calcium-phosphorus solution was continuously injected into the capillary, and the temperature was kept at 36.5±0.5° C., and deposited for 14 days to prepare the capillary coated with hydroxyapatite.

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Abstract

A stationary phase for ion-exchange chromatography is provided. The stationary phase comprises a chromatographic matrix superficially coated with hydroxyapatite. The stationary phase for ion-exchange chromatography has good biocompatibility and bioaffinity and higher mechanical strength. The stationary phase for ion-exchange chromatography is prepared by the following steps: activating an inorganic chromatographic matrix material such as silica gel or Zr / Mg composite oxide, immersing the matrix material into an oversaturated calcium / phosphorus solution, standing for deposition at 36.5 plus or minus 0.5 DEG C to obtain hydroxyapatite coating, washing with water twice, and drying to obtain the hydroxyapatite-coated stationary phase for ion-exchange chromatography in the protein separation. The method is also used to prepare a coating on the inner wall of a capillary tube to reduce the non-specific adsorption of proteins and simultaneously to provide an ion-exchange function for improvement of separation selectivity.

Description

technical field [0001] The invention relates to an ion-exchange chromatographic stationary phase, a preparation method and application thereof. Background technique [0002] Ion exchange is an essential separation mode in high performance liquid chromatography (HPLC), which is widely used in biomedical fields, such as amino acid analysis, separation of peptides and proteins. When using ion exchange chromatography to separate proteins, the charge density and isoelectric point value of the separated protein and the ion capacity of the ion exchanger on the chromatographic column determine the strength of the retention capacity. [0003] Ion-exchange chromatography stationary phases are roughly divided into two categories: organic and inorganic matrices according to the composition of their matrices. Organic matrix fillers have good chemical stability, can be used in a wide range of pH values, are resistant to alkali washing, and have good regeneration capabilities. However, t...

Claims

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Application Information

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IPC IPC(8): B01J20/282B01J20/32B01J43/00
Inventor 冯钰锜李烃
Owner WUHAN UNIV
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