Method Conditions:
Voltage: 10 kV
Capillary: MicroSolvCE Bare Fused Silica 75µm 39cm Total, 8.5cm Effective
Injection: Hydrodynamic, 7mbar, 5 seconds
Run Buffer: CElixir Accelerator (B) Solutions Mixed to pH 7.0
Organic Additive: None
Detection: UV 214nm

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Using a Micro-analytical CE-211 Series Capillary Electrophoresis System (MO, USA) and a MicroSolvCE 75µ bare fused silica capillary, the separation of Cytochrome C and an impurity was easily accomplished. Mixing two different CElixir Accelerator Solutions (pH 6.2 and 8.2), the resulting BGE/run buffer pH was 7.0. The Cytochrome C sample (0.5 mg/ml) was dissolved in the CElixir™ pH 7.0 solution and injected directly onto the capillary after it was conditioned according to the CElixir™ Operating and Trouble Shooting Manual. A 5 second injection was made. The separation was completed within five minutes after injection.

Discussion and Rationale:
The pH of the BGE/run buffer was adjusted to 7.0 because at this pH (a very common biological pH), the Cytochrome C would be positively charged and this pH allows for good solubility of the protein and the associated impurity. The first peak in the electropherogram is Cytochrome C, the second is an impurity and the third is a neutral marker.

When the sample was analyzed in 25mM phosphate pH 7.0 buffer and injected onto the same type of capillary with the same conditions, no peak could be seen. Increasing the strength to 100mM phosphate pH 7.0, a very broad peak (5000 Theoretical Plates) could be seen. With CElixir™ the above electropherograms can be seen.

MicroSolv Technology Corporation would like to thank Dr. Wang of Peking University for this application.
Dr. Tiansong Wang
Professor of Chemistry
Peking University
Beijing, China

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