Chemical synthesis of peptides, and even proteins, offers the possibility to expand the functionality and stability imbued by nature. However, chemical synthesis of very long peptides and small proteins remains today an exceedingly difficult task. Several ligation strategies have been developed that help to alleviate this challenge. These strategies though, require a purified, yet fully protected peptide fragment.
Purification of a fully protected peptide species can be challenging by standard reversed-phase techniques, primarily due to the limited solubility of protected peptides in aqueous solutions. In today’s post, I will discuss using normal-phase chromatography for purification of protected peptides.
Continue reading Can you use normal-phase chromatography to purify protected peptides?
Every now and again I hear the question “which solvent do you recommend for my solid phase peptide synthesis?” Historically, dichloromethane (DCM) was used as a solvent for solid phase synthesis as the kinetics of amino acid activation and amine coupling were much more favorable. However, solubility concerns, particularly for Fmoc-protected amino acids limited the utility of the solvent. Nowadays, DMF and NMP are the two principle solvents for both microwave assisted and room temperature solid phase peptide synthesis. But the question remains, which one is better?
In today’s post, I will compare how the choice of dimethylformamide (DMF) or N-methylpyrolidone (NMP) effects the synthesis of a short yet very hydrophobic peptide.
Continue reading Synthesizing hydrophobic peptides – choosing the right solvent
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