How to purify synthetic peptides: what are the options?

Would you ever consider an alternative to reversed- phase HPLC to purify your synthetic peptides?  It seems like a silly question, right.  And like many of you, I literally laughed at my Product Manager when he asked me this same question in my first days at Biotage.

Fast forward a few years and my answer to that question is now very different.  For those of you that have followed this blog, you’ll know that I have switched to reversed-phase flash chromatography almost exclusively for my peptide purification.  In today’s post, I’ll highlight some of the critical reasons that have influenced my change in mindset.

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How does flow rate affect my peptide purification efficiency when using a small pore stationary phase?

In a previous post, I evaluated how flow rate can impact my purification efficiency using flash chromatography.  I noticed though, at high flow rates a significantly later elution time for my peptide.  I hypothesized that the increased pressure was driving the compound further into the pores, increasing the overall interaction with the stationary phase and causing the increased retention.  We know that the particle size and particle pore size impact resolution and purification efficiency, so how does flow rate play a role with a different stationary phase?

In today’s post I’ll evaluate several flow rates using a reversed phase stationary phase material with slightly larger diameter particles that possess significantly smaller pores.  The smaller pores should limit the access of the peptides to the stationary phase and negatively impact the purification.

Continue reading How does flow rate affect my peptide purification efficiency when using a small pore stationary phase?