Almost all the peptides I have synthesized were subsequently purified using a reversed-phase C18 column.  Sometimes this worked, but sometimes it didn’t work so well.  When my C18 purifications failed, I questioned whether or not I could have predicted this outcome prior to extensive HPLC efforts.  Since then, I have learned that the amino acid composition of the peptide may give some clues to the peptide’s chromatographic behavior.

While there are numerous stationary phase functionalization types for reversed-phase chromatography, in today’s post I will describe some differences I have observed when purifying peptides using C18- or C4- functionalized stationary phases for peptide purification.

In a previous post, I evaluated how flow rate can impact my purification efficiency using flash chromatography.  I noticed though, that my peptide eluted significantly later with high mobile phase flow rates.  I hypothesized that the increased pressure (caused by higher flow rates) 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?

Resins for solid phase peptide synthesis can vary significantly in both functionalization and composition, leading to mixed results at the end of a synthesis.  Previously, I demonstrated how the resin loading level affects the success or failure of your peptide synthesis.

In today’s post, I’ll highlight how both the hydrophilicity and swelling capacity of your resin can influence your peptide synthesis.

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 contains 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.

It used to be easy with only polystyrene based resin types, but nowadays there is a broad choice of types to choose from, including everything from the C-terminal functionality (Rink vs Wang) to the polymer from which the resin itself is synthesized.

All resins have one thing in common, and that’s the reactive site loading level. In this post, I will share my experiences with how this important factor impacts the success of peptide synthesis.

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.

More and more groups are exploring the utility of peptides with an ever widening variety of applications. And although peptides are getting cheaper to purchase outright, many groups are continuing to bring peptide synthesis in house. As more groups join the peptide community, I frequently encounter questions about the basics of peptide synthesis.

As a chemist new to the peptide community, there are many choices that have to be made.  Which coupling reagents to use? Heat or no heat to promote chemistry? And most importantly, which resin?  I have talked previously about resin choices, from loading levels to swelling capacity and how they affect the synthesis outcome.  But I haven't addressed yet a fundamental feature of commercially available resins, and that's the functional handle to which the peptide chain is conjugated.

In today's post, I'll describe some, and I mean only some, of the most commonly used chemical functionalities for Fmoc-based solid phase peptide synthesis and some scenarios in which you would choose one resin type over another.

While resins loaded with the natural 20 amino acids are commercially available these days, there may be times when loading the first amino acid onto the resin in house may be necessary.  And unlike loading the first amino acid onto amide-leaving resins, the first coupling reaction for C-terminal acids can be chemically more challenging.

While many of the standard amino acids can be purchased pre-loaded onto Wang type resins, there are still cases where coupling the first amino acid onto Wang resin manually is necessary.  In my case, an unnatural amino acid was required on the C-terminus so there was not a commercially available source.