High Performance Size Separation of Very High Molecular Weight Proteins
Size separation of high molecular weight proteins by Gel Filtration Chromatography (GFC) is a widely used technique.
Typical size exclusion products have good protein resolution due to high pore volume but this leads to low mechanical strength and a maximum operating pressure of 1000 psi. The particle size is typically limited to 8 mm to reduce backpressure but this imposes a limit on the speed of analysis and the ability to run multiple columns.
Exsil™ GFC 5000 has been developed to complement the GFC 3000 and GFC 4000 ranges and provide a high efficiency product for the separation of very high molecular weight proteins with superior bed stability at an economical price.
- Phase: Stable High Polarity (pH 2-7.5)
- Base Silica: Exsil™ 5000S, high purity silica
- Particle Size: 5 µm
- Pore Size: 800A
- Pore Volume: 1.2 cc/g
- Exceptional Stability: Can be packed at 7000 psi.
- High Efficiency: Up to 100,000 plates/m for 8mm id
- High Recovery: No evidence of protein loss.
- Wide Applicability: Low adsorption of basic proteins
- Cost Effective: High performance at low cost.
- Reproducible: Typical Exmere reliability
Size Exclusion Range
Gel Filtration Chromatography of proteins shows that the Exsil GFC 5000 phase shifts the retention of proteins to higher Kd values compared to the Exsil GFC 3000 and GFC 4000 phases.
The optimum operating range of the GFC 5000 phase is 150 K – 1250K Daltons which is significantly higher than the 50 K- 500K of the GFC4000 making it more suitable for very high molecular weight proteins.
Together the three phases (GFC3000,4000 and 5000) cover the full range from 15 – 1000K.
The large pore size of the Exsil GFC5000 phase allows the resolution of the high molecular weight Thyroglobulin (MW 670,000) which is partially excluded on smaller pore media.
High Speed and High Resolution Separations
Exsil GFC 5000 retains the excellent mecahnical stability of the Exsil GFC 3000 phase and can be packed at 7000 psi and operated at up to at least 3000 psi.
This high stability extends the opportunity to carry out either high speed or high resolution separations to very high molecular weight proteins.
The chromatograms show a high speed separation completed on a 250 mm column in 3 minutes and a high resolution separation using three 250 mm columns. In both cases the back pressure was in the region of 2000/2500 psi which is well within the operating capabilities of the Exsil GFC5000 phase but twice the maximum limit of some competitive materials.
Protein Recovery/ Phase Clean up
Based on the same bonding as the Exsil GFC3000/4000 series, similar high protein recoveries combined with reistance to high pH cleaning can be expected.
Exsil GFC5000 offers a robust, high efficiency phase for the separation of very high molecular weight proteins and provides the ideal complement to the Exsil GFC3000 and GFC4000 series.