Synthesizing hydrophilic peptides can be relatively straightforward, especially when compared to the trials and tribulations encountered when synthesizing hydrophobic peptides. However, purifying hydrophilic peptides using standard reversed-phase chromatography can be a challenge. In a previous post, I encountered this problem during my analytical HPLC analysis of crude peptide mixtures.
In this post, I will discuss the use of ion pairing agents to increase the perceived hydrophobicity of your peptide, increasing the column retention and enabling a smooth purification of your hydrophilic peptide.
Continue reading How to purify hydrophilic peptides with flash column chromatography
Historically, solid phase peptide synthesis has been conducted at room temperature, demanding long reaction times and often double coupling to ensure a quality crude peptide product. More recently however, different strategies have been identified to heat the reactor vial, increasing the overall reaction rate and potentially the crude purity of your peptide.
In today’s post I will demonstrate that microwave heating can improve the crude purity of your desired peptide.
Continue reading Microwave heating – a route to better quality crude peptides
In previous posts I have described using high concentrations of amino acids to improve your peptide synthesis among some other tips and tricks. But there is a particularly handy tip that was left off the list.
Weighing out and dissolving the amino acids and coupling reagents requires the greatest amount of manual effort when setting up a peptide synthesis, particularly for automated instruments. One way to alleviate some of this time investment is to generate stock solutions of your amino acids for use over the course of several syntheses. You’re probably asking yourself though: “how long are those amino acid solutions actually stable?”
In today’s post I’ll answer that question by comparing the crude purity of peptides synthesized using amino acid stock solutions or freshly dissolved amino acids.
Continue reading How long are amino acids stable in solution?
There are several strategies employed when a peptide synthesis requires optimization. Typically, the first thing considered is whether or not to double couple specific amino acids within the sequence. This is somewhat of a change in mentality from traditional room temperature synthesis strategies where double coupling is frequently used for the entire peptide sequence.
In a previous post, I briefly described several scenarios in which doubling coupling can be used in conjunction with microwave heating to improve the overall crude peptide purity. In today’s post, I will delve more deeply into the question of whether or not double coupling is necessary to improve your peptide synthesis.
Continue reading Using double coupling to improve your peptide synthesis
It used to be easy with only polystyrene based 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.
Continue reading How do I choose my resin for peptide synthesis? Part 1
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