High Performance Size Separation of High Molecular Weight Proteins
Exsil GFC 4000 has been developed to provide a high efficiency product with superior bed stability at an economical price.
- Phase: Stable High Polarity (pH 2-7.5)
- Base Silica: Exsil 4000S, high purity silica
- Particle Size: 5 µm
- Pore Size: 400A
- Pore Volume: 1.2 cc/g
- High Stability: Can be packed at 7000 psi.
- High Efficiency: Up to 100,000 plates/m for 5mm 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 4000 shifts the retention of proteins to higher Kd values compared to the Exsil GFC 3000. The Kd for albumin is in the region of 0.68 which compares well with the reported figure for TSK 4000SW of 0.70.
The optimum operating range of the GFC 4000 is shifted to 60 K – 500K which is significantly higher than the 15 K- 60K of the GFC3000.
Together the two columns cover the important 15 – 500K region.
Exsil GFC4000 produces excellent resolution both in standard 8mm id columns and in higher sensitivity 4.6 mm format.
High Speed and High Resolution Separations
Exsil GFC 4000 retains the excellent mecahnical stability of the Exsil GFC 3000 and can be packed at 7000 psi and operated at up to at least 3000 psi.
This high stability opens up the opportunity to carry out either high speed or high resolution separations. 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 psi which is well within the operating capabilities of Exsil GFC4000 but twice the maximum limit of some competitive materials.
Recovery was assessed using the acidic protein, Human Serum Albumin and the basic protein, Equine Cytochrome C using 50mM pH 7 potassium phosphate and 150 mM sodium chloride eluent. In neither case was there evidence of protein loss with recoveries in the 99 to 102% range.
Although the recommended working pH is 2 – 7.5, cleaning the column with 50 column volumes of 50% 10mM NaOH/50% MeOH had minimal effect on the column performance as is shown in the following chromatograms.
If the clean-up step is restricted to 5 column volumes, then the column could be cleaned a minimum of 10 times. However, the low apparent protein adsorption would indicate that, provided samples and eluent are filtered, column clean-up should only be required infrequently.
Exsil GFC4000 offers a robust, high efficiency phase for the separation of high molecular weight proteins.