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 functionalizations 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 purifications.
Continue reading Which stationary phase should I chose for my peptide purification?
Conversations are routinely held regarding handling hydrophobic peptides, but hydrophilic peptides offer their own challenges when it comes to purification. In a previous post, I synthesized Octa-Arg, an extremely hydrophilic peptide. I used ion pairing reagents to increase the peptide’s overall retention by the stationary phase, but choosing the solvent should to use for solubilizing the peptide for purification by flash column chromatography was no easy task.
In today’s post, I’ll investigate several solvents commonly used to inject peptide samples for purification and evaluate their impact in peptide retention by the stationary phase.
Continue reading Why won’t my peptide stick to my column?
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