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?
Have you ever wondered if there was a faster and cheaper way to purify your peptides?
My colleagues and I in the peptide community rely almost exclusively on reversed-phase HPLC for delivery of highly pure peptide products. However, this process is often very time consuming and requires expensive columns and solvents to be successful. Alternatively, peptide purification via reversed-phase flash column chromatography can be used to complete a purification in a fraction of the time and with a fraction of the costs.
Here I will show how I do gradient optimization for peptide purification via reversed-phase flash column chromatography and will highlight the similarities with standard HPLC methodologies.
Continue reading Optimizing a mobile phase gradient for peptide purification using flash column chromatography
Peptides, by nature, are composed of amino acids with potentially ionizable chemical moieties. The ionization state of any of these moieties can significantly impact the peptide’s chromatographic behavior, both in terms of peak shape and retention by the solid support. Peptide purification by reversed-phase chromatography, however, almost exclusively includes an acidic additive to the mobile phase solvents, maintaining the solution at a pH of 2-3 throughout the purification cycle. But have you ever considered trying an alternative additive in the mobile phase to improve your purification results?
In the following post I discuss the impact of mobile phase pH in the purification of oxytocin (CYIQNCPLG-NH2), a 9-amino acid peptide that requires disulfide bond-mediated cyclization for its biological activity.
Continue reading Peptide purification improvements with flash column chromatography by modulating mobile phase pH
When it comes to synthesizing a peptide, the first thing that comes to mind is the number of stoichiometric equivalents to use. Sometimes that number is as few as 1.5, sometimes it’s as high as 20!
But have you ever thought about the liquid volume that contain those molecules and how that might affect the success of your coupling reaction? In this post I will discuss the impact of amino acid concentration in the overall success of solid phase peptide synthesis.
Continue reading Does amino acid concentration really matter during peptide synthesis?