Sample displacement chromatography (SDC) is normally associated with purification of macromolecules but has been employed for peptide purification for many decades. The technique involves overloading of a sample mixture onto a purification column, exploiting differences in affinity of the product/impurity mixture for the stationary phase. As the sample is loaded, the species with high affinity displace those with a lower affinity for the media as the components compete for available binding sites, thus separating the mixture. However, as SDC is low pressure, it typically results in enriched bands of products, rather than sharp baseline resolved peaks.
SDC was superseded by the invention of technology that allowed scale up of high-pressure chromatographic (HPLC) methods, giving higher fraction purity by improved product resolution. However, recent advances in the quality of media for low pressure purification (flash) has enabled SDC of peptides to be revived as a complementary method to HPLC.
SDC is optimally performed in highly overloading conditions and utilizes the full binding site capacity of the media. Therefore, the advantage of the SDC technique over reverse phase prep HPLC is high throughput with lower material costs, lower waste and lower hardware costs. This allows for accessible scale up using simple labware, such as cartridge columns, flash LC systems and peristaltic pumps.
We will show that SDC can be used to purify short to medium length peptides (~10-25 AA) to a high purity using benchtop hardware, with a significant reduction in solvent use and waste. We will also demonstrate our ability to predict the capacity and product distribution throughout the purification media.
