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Biomolecular Separations:
Lactalbumin, Trypsin Inhibitor, Bovine Albumin and Carbonic Anhydrase Mixture
Method Conditions:
Voltage: 28 kV
Capillary: MicroSolvCE Bare Fused Silica 25µm 27cm long
Injection: Hydrodynamic
Run Buffer: CElixir Accelerator Solution, pH 6.2, 7.2, 8.2 and 9.2
Organic Additive: None
Detection: 190nm UV |
Click here for catalog description of CElixir
Method:
Using a Beckman P/ACE 5000 capillary electrophoresis instrument and a bare fused silica capillary, the
separation of a protein mixture was found. A solution of Bovine Albumin, Lactalbulin, Trypsin Inhibitor and Carbonic
Anhydrase was prepared using good laboratory practice. Four different CElixir pH’s were used to “scout” for the
method that showed the most separation. Ramping the voltage for one minute before beginning the separation was
employed after inserting a 1 sec injection of a “water plug”. This aided in the precision of this method.
Discussion and Rationale:
The first run that was performed was done in the “borate” CElixir at pH 9.2 without sufficient. Since
CElixir does not suffer from pH hysteresis when changing pH, the same capillary was used without any
conditioning steps. CElixir pH 8.2 was then run without sufficient resolution. The third run was performed by
using CElixir pH 6.2 and resolution was achieved.
A fourth run was preformed by adding together CElixir Accelerator solutions of pH 6.2 and pH 8.2 (according
the CElixir Manual) that resulted in an CElixir of pH 7.2. This run did not produce any better resolution but
indicates that with more pH optimization, the first two peaks may show baseline resolution.
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