Gel filtration chromatography is a technique for elution and separation according to the difference of molecular weight of each component in the separated sample by using the action of molecular sieve of gel particles with porous network structure. Today we mainly introduce some important parameters in gel filtration chromatography.
Parameters of gel
1. Gel particle size
The size of the particle is usually expressed in mesh or diameter (μm). Gels used for chromatographic separation are generally made into spherical particles. The size of gel particles is directly related to the flow rate and resolution of the column. The larger the gel particles are, the faster the flow rate is, but the resolution is poor. Conversely, the smaller the particles, the slower the flow rate, but the higher the resolution.
2. Limit of discharge resistance
Refers to the molecular weight of the smallest molecule that cannot enter the internal pores of the gel particle. The exhaust resistance limit reflects the maximum molecular weight of a gel that can be effectively separated. All molecules greater than the exhaust resistance limit are completely blocked and cannot be separated. For example, the exhaust resistance limit of Tandex G25 is 5000Da, which indicates that molecules with molecular weight greater than 5000Da are total exhaust resistance.
3. Grading area (or work area)
The molecular weight range of the largest and smallest molecules that can enter the internal pores of the gel. It represents the separation range applicable to a gel. For example, Tandex G25 has a grading separation range of 1000-5000Da for spherical proteins, which means that Tandex G25 can effectively separate spherical proteins with molecular weight within this range.
4. Water absorption capacity
1g of the volume or mass of water absorbed by dry glue. The bigger the gel, the more water it absorbs.
5. Volume of bed
The final volume of 1g dry glue after absorbing water.
Volume parameter
1. External water volume
The sum of the void volumes between the gel particles in the column, expressed as Vo.
2. Matrix volume
The volume inherent in the particle itself after the swelling of the dry glue is expressed by Vg.
3. Internal water volume
The sum of the pore volume inside the gel particles, or the sum of the volume that small molecules of solutes can penetrate into the gel interior, also known as the stationary phase volume, is represented by Vi.
4. post bed volume
The total volume from the bottom plate of the gel column to the surface of the gel deposit, also known as the bed volume, is expressed by Vt.
The calculation formula is as follows:
Since Vg is relatively small and can be ignored, it can be written as follows:
5. Elution volume
The volume of eluent required for a separated sample to be completely eluted from the gel column is indicated by Ve.
The calculation formula is as follows:
Where, Kd is the distribution coefficient; Ve is generally between Vo and Vo+Vi. For large molecules with complete discharge resistance, because they do not enter the gel interior, they can only flow in the particle gap, so the elution volume Ve=Vo. For fully permeable small molecules, the elution volume Ve=Vo+Vi is obtained because it can flow in the whole gel column (Vg is ignored). Molecules with molecular weights in between have elution volumes in between. Some adsorption proteins are washed out at the end.
Welch gel filtration packing materials
- Tandex G series and LH20 multi-mode gel filtration series
- SuperTandex Prep Grade series
- Tanrose Fast Flow series
- Tanrose/Tanrose CL series
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