Packing method and detecting method of gel chromatography column

A gel chromatography column and column packing technology, applied in the field of chromatography, can solve the problems of toxic reagents, volatile, product quality and safety impact, etc., and achieve the effect of improving column packing efficiency

Active Publication Date: 2014-01-01
SINOVAC BIOTECH
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  • 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

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

Example Embodiment

[0035] Example 1:

[0036] The gel is Sephacryl S-400HR. produced by GE. See the process figure 1 .

[0037] 1) Preparation of gel suspension: use 0.02mol / L NaOH solution and 0.01mol / L PBS (containing 0.05mol / L NaCl, pH7.2) solution 1:1 to mix the alkali salt buffer solution for homogenization, gel and alkali salt The ratio of buffer is 1:1 to obtain a gel suspension

[0038] 2) Packing the column: add purified water with a height of 1cm at the bottom of the column tube, then pack the column with gel suspension, pack the column head of the chromatography column, and pump the 0.02mol / L NaOH solution with the constant pressure of 1.0bar. 0.01mol / L PBS (containing 0.05mol / L NaCl, pH 7.2) solution 1:1 mixed alkali salt solution, when the gel height is constant at constant pressure, the prepared gel column is 65cm high; proceed to the next step;

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

Example Embodiment

[0046] Example 2

[0047] 1) Preparation of gel suspension: mix 0.4mol / L NaOH solution and 0.01mol / L PBS (containing 0.8mol / L NaCl, pH 7.4) solution 1:3 for homogenization; the ratio of gel to alkali-salt-alkali-salt buffer solution 1:3; get a gel suspension

[0048]2) Column loading: add purified water with a height of 2cm at the bottom of the column tube, load the gel suspension, install the column head, and then pump 0.4mol / L NaOH solution and 0.01mol / L PBS (containing 0.8mol / L NaCl, pH7.4) solution 1:3 mixed alkali-salt buffer to prepare gel column, the constant pressure is 1.0bar to the same height, and the prepared gel column is 70cm high;

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

[0050] 4) Column efficiency detection:

[0051] a) Acetone control: use purified water to prepare 2% acetone solution as a tracer, the loading volume is 4% of the colum...

Example Embodiment

[0056] Example 3

[0057] 1) Preparation of gel suspension: mix 0.2mol / L NaOH solution and 0.01mol / L PBS (containing 0.3mol / L NaCl, pH 7.3) solution 1:1.5 for homogenization, and the ratio of gel to alkali-salt buffer is 1:1. 1.5; obtain a gel suspension;

[0058] 2) Column loading: add purified water with a height of 4cm at the bottom of the column tube, load the gel suspension, install the column head, and pump 0.2mol / L NaOH solution and 0.01mol / L PBS (containing 0.3mol / L NaCl, pH7.3) solution 1:1.5 mixed alkali salt solution to prepare gel column, the constant pressure is 1.0bar to the same height, 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 / L NaCl, pH 7.3) buffer, and the equilibrium flow rate is 34cm / h;

[0060] 4) Column efficiency detection:

[0061] a) Acetone control: use purified water to prepare 2% acetone solution as a tracer, the sample volume is 2% of the column ...

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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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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
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