This document introduces a protein separation method based on high-performance liquid chromatography. The method uses non-covalent affinity chromatography columns and polyacrylamide gel chromatography columns, which can effectively separate proteins of different molecular weights.
This document is an application brief by Robert D. Ricker from Agilent Technologies regarding size-exclusion chromatography (SEC). It discusses the use of standard non-denaturing mobile phases versus denaturing mobile phases containing guanidine hydrochloride (GuCl) or SDS. The text explains that denaturing phases are used to ensure un-aggregated monomers for proper size determination, noting that this shifts molecules toward larger apparent sizes and smaller retention volumes.
| Molecular-weight range | 4,000-400,000 daltons |
|---|---|
| Column dimensions | 9.4 x 250 mm |
| Part Number | 884973-901 |
| Detection wavelength | 254 nm |
| Flow rate | 2.0 mL/min |
| Injection volume | 20 uL |
Denaturing mobile phases containing guanidine hydrochloride or SDS are used to insure un-aggregated, monomers for proper size determination.
Denaturation shifts molecules toward larger apparent size and smaller retention volume.