When it comes to the purification of polar, water-soluble compounds reversed-phase chromatography is the most commonly used approach. However, because of strong stationary phase – mobile phase repulsion forces, the use of highly aqueous (90-100% water) solvent systems has been shown to provide less retention than needed. This issue has led to the development of “aqueous compatible” reversed-phase media.
In this post I explore if these types of phases are actually needed by looking at the separation of some very polar and low log P compounds using a “traditional” C18-bonded silica.
Continue reading Are reversed-phase flash chromatography columns designed for aqueous solvents necessary?
Purification by reversed-phase chromatography relies primarily on a hydrophobic interaction of the molecule with the alkyl chains bonded to the stationary phase for column retention and elution through a partitioning mechanism. While this is certainly true for purification of peptides, surface area accessibility and media particle size also play critical roles in the resolving power of a particular stationary phase. The particle size influences the loading capacity, however pore size greatly influences molecular accessibility and therefore resolving power.
In today’s post, I will demonstrate how pore size can impact your peptide purification using flash column chromatography.
Continue reading How does media pore size impact peptide resolving power?