Molecular exclusion chromatography, also known as gel chromatography, is the use of different molecular sizes of the separated substances lead to different degrees of penetration in the filler so that the separation of components; commonly used filler molecular sieve, dextran gel, microporous polymers, microporous silica or glass beads, etc., according to the nature of the stationary phase and the test material as the mobile phase of the choice of water or organic solvents.

Operation method

basic program

Materials and Instruments

Protein
SE Buffer
Chromatography column

Move

1. Wash the SE column with degassed HPLC-grade water to remove the storage solvent at a flow rate of 1 ml/min. Equilibrate the SE column with degassed SE buffer at a flow rate of 1 ml/min for 30 min at room temperature or until the baseline is stable.

2. Inject 100 μl of buffer to perform a pseudo-chromatography of a blank sample with the detector set at 210-220 nm, 0.1-1.0 AUFS. Suitable settings are typically 0.1 AUFS at 100 pmol, 0.3 at 500 pmol, 0.5 at 1 nmol, and 1.0 at 2 nmol; the plotting speed is 0.5 cm/min.

3. Centrifuge the protein standard or protein mixture at 2 000 g for 5 min and inject 100 μl of supernatant and repeat step 2. Collect each chromatographic peak in a different polypropylene tube and determine the elution volume of the peak.
4. The divisional components are desalted and the solvent is removed with a Speedvac evaporator.5. The retention time of the last eluted peak should not be more than twice that of the first peak, if this is not the case the mobile phase is spiked with 20% methanol to inhibit protein binding to the medium.

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