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Packing method and detecting method of gel chromatography column

Active Publication Date: 2014-12-10
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The column efficiency detection is to detect the system after the column is packed. The method of measuring the column efficiency uses acetone as a tracer for the column efficiency detection, but acetone is a toxic reagent and is volatile, which has a certain impact on the experimental operators; at the same time, acetone It is easy to remain in the column, and it is difficult to completely remove it in the subsequent process, which has a certain impact on the quality and safety of the product

Method used

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  • Packing method and detecting method of gel chromatography column
  • Packing method and detecting method of gel chromatography column
  • Packing method and detecting method of gel chromatography column

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The gel was Sephacryl S-400HR produced by GE Company. see process figure 1 .

[0037] 1) Preparation of gel suspension: use 0.02mol / L NaOH solution and 0.01mol / LPBS (containing 0.05mol / LNaCl, pH7.2) solution 1:1 mixed alkali salt buffer solution to homogenize the gel, gel and alkali salt The ratio of buffer is 1:1 to get a gel suspension

[0038] 2) Column packing: Add purified water with a liquid level of 1cm at the bottom of the column tube, then fill the column with gel suspension, install the column head of the chromatography column, and pump 0.02mol / L NaOH solution with a constant pressure of 1.0bar 0.01mol / LPBS (containing 0.05mol / L NaCl, pH7.2) solution 1:1 mixed alkali-salt solution, constant pressure until the gel height remains constant, the prepared gel column height is 65cm; proceed to the next step;

[0039] 3) Equilibration: Equilibrate 1 times the column volume with 0.01mol / L PBS (containing 0.05mol / L NaCl, pH7.2) buffer solution, and the equilibrium f...

Embodiment 2

[0047] 1) Preparation of gel suspension: Mix 0.4mol / L NaOH solution and 0.01mol / LPBS (containing 0.8mol / L NaCl, pH7.4) solution 1:3 to homogenize the gel; the ratio of gel to alkali salt buffer solution 1:3; get a gel suspension

[0048]2) Column packing: add purified water with a liquid level of 2cm at the bottom of the column tube, fill in the gel suspension, install the column head, and then pump in 0.4mol / L NaOH solution and 0.01mol / L PBS (containing 0.8mol / L NaCl, pH 7.4) solution 1:3 mixed alkali-salt buffer to prepare a gel column, the constant pressure is 1.0bar to the same height, and the prepared gel column is 70cm high;

[0049] 3) Equilibration: equilibrate 3 times the column volume with 0.01mol / L PBS (containing 0.8mol / LNaCl, pH7.4) buffer solution, and the equilibrium flow rate is 35cm / h;

[0050] 4) Column efficiency test:

[0051] a) Acetone control: 2% acetone solution was prepared with purified water as a tracer, the sample volume was 4% of the column volum...

Embodiment 3

[0057] 1) Preparation of gel suspension: mix 0.2mol / L NaOH solution and 0.01mol / LPBS (containing 0.3mol / L NaCl, pH7.3) solution 1:1.5 to homogenize the gel, the ratio of gel to alkali salt buffer is 1: 1.5; Obtain gel suspension;

[0058] 2) Packing: Add purified water with a liquid level of 4cm at the bottom of the column tube, fill in the gel suspension, install the column head, and pump in 0.2mol / L NaOH solution and 0.01mol / LPBS (containing 0.3mol / L NaCl, pH 7.3) solution 1:1.5 mixed alkali-salt solution to prepare a gel column, the constant pressure is 1.0bar to the height unchanged, and the prepared gel column is 72cm high;

[0059] 3) Equilibration: Equilibrate 1.5 times the column volume with 0.01mol / L PBS (containing 0.3mol / LNaCl, pH7.3) buffer solution, and the equilibrium flow rate is 34cm / h;

[0060] 4) Column efficiency test:

[0061] a) Acetone control: 2% acetone solution was prepared with purified water as a tracer, the loading volume was 2% of the column volu...

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Abstract

The invention provides a packing method of a gel chromatography column. The packing method comprises the following steps: 1), preparing a gel suspending solution, namely, adding a 0.02-0.4mol / L NaOH solution and a phosphate buffer solution containing NaCl in gel, to form an alkali and salt mixed gel suspending solution; 2), packing, namely, adding purified water into the bottom of a column tube, and filling the gel suspending solution into the column tube; pumping the alkali and salt buffer solution in which the NaOH solution and the phosphate buffer solution containing NaCl are mixed according to the ratio of 1: (1.0-3) into the column tube, and starting the next balancing step when the level of the gel suspending solution is constant; 3), balancing, namely, balancing 1-3 times of column volume by using the buffer solution containing NaCl; and 4), column effect detecting, namely, with the NaCl as a tracer agent, monitoring the change of conductivity, so as to obtain a column effect result. Compared with the traditional packing method, the packing method provided by the invention has the packing effect same as that of the traditional method, furthermore, by adoption of the packing method provided by the invention, the packing efficiency can be greatly improved, and the disadvantages of repeat packing and large balancing volume are overcome.

Description

technical field [0001] The invention belongs to the technical field of chromatography, and in particular relates to a preparation method and detection of a chromatography exchange column. Background technique [0002] Gel chromatography, also known as gel filtration, is a chromatographic method for separating substances by molecular weight. In this method, the sample is added to a chromatography column filled with gel particles, and then eluted with a buffer. Large molecules cannot enter the stationary phase in the gel particles, but only stay in the mobile phase between the gel particles, so they flow out of the chromatography column at a faster speed, while small molecules can freely enter and exit the gel particles. And quickly form a dynamic equilibrium between the mobile phase and the stationary phase, so it takes a long time to flow through the column bed, so that molecules of different sizes can be separated. [0003] Gel chromatography is a fast and simple separati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N30/06G01N30/02
Inventor 张健张颖赵金玉董兴华马敏华李静尹卫东
Owner SINOVAC BIOTECH