Chromatography principles
What determines a successful purification scheme?
The output of a protein purification scheme is traditionally described in terms of purity, homogeneity, and yield. Often, optimization of one of the output parameters can only be achieved at the expense of the other output parameters, and each purification step will therefore be a compromise.
Purity:
- Measured by electrophoresis, specified as target protein-to-total protein ratio.
- Measured by size exclusion chromatography.
Homogeneity:
- Measured by size exclusion chromatography, specified as monomer-to-aggregate content.
- Measured by native electrophoresis.
Yield:
- Measured by absorption at 280 nm and using the extinction coefficient for the target protein.
- Measured by activity assay, specified as U/L.
What phases constitute a chromatographic step?
Each chromatographic step can be split into several phases, such as sample load, wash, and elution. Each phase can involve different factors (such as pH, conductivity, additives, and flow rate), of which all can have a profound effect on the outcome of the chromatographic step.
What chromatography methods can I choose from?
Chromatographic methods separate according to differences between the properties of the protein to be purified (the target protein) and the properties of other substances in the sample.
| Protein property |
Method |
| Specific ligand recognition | Affinity chromatography (AC) |
| Metal ion binding | Immobilized metal ion affinity chromatography (IMAC) |
| Charge | Ion exchange chromatography (IEX) |
| Size | Size exclusion chromatography (SEC) |
| Hydrophobicity | Hydrophobic interaction chromatography (HIC) Reversed phase chromatography (RPC) |
| Combination of several protein properties, such as charge, size or hydrophobicity |
Multimodal chromatography (MM) |
How do I select a suitable chromatography resin?
Purification efficiency is highly dependent of the chromatography resin selected for each technique. The efficiency, flow resistance, selectivity, and binding capacity differ between resins. The particle size of the resin strongly affects efficiency and flow resistance. A resin with large beads gives chromatography columns with low resolution (broad peaks) but generates low backpressure. Small beads give higher resolution (narrow peaks) but also generate higher backpressure.
A general principle of choosing chromatography resins is a larger bead size for early purification steps, and smaller bead size for later steps, where demand on purity is increased.