How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?

Recently there has been substantial motivation to consider and evaluate alternative, more environmentally friendly solvents.  Some countries have even gone so far as to ban some of the more toxic, yet commonly used solvents.  In addition to general toxicity, additional consideration in the green chemistry movement is the volume of solvent used in any particular application.  In this regard, purification solvent selection is closely monitored as they are often used in large quantities.

One alternative that is growing in popularity is the use of methanol in place of acetonitrile for reversed phase purification of small molecules.  Methanol is certainly less expensive, but is also a more environmentally-friendly solvent for use in purification applications.  But it’s use for peptide purification has not been widely adopted to date.  In today’s post, I’ll compare the purification efficiency of methanol when compared to acetonitrile for peptide purification by reversed phase flash chromatography.

Continue reading How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?

How to purify peptides using a step gradient in flash column chromatography

Flash chromatography can be a challenging technique for peptide purification due to the lower resolution achieved with large particles.  While some may see this as a disadvantage, the significantly greater loading capacity gives me reason to make this work. So how can I achieve the high purity levels often accessed using traditional reversed-phase HPLC methods?

In this post, I’ll discuss using a step gradient for peptide purification.  Step gradients are commonly used in normal-phase small molecule purification and typically improve the purification efficiency while reducing the overall purification time. Continue reading How to purify peptides using a step gradient in flash column chromatography

How long should I let my cleavage reaction stir at room temperature?

As the rules for cell permeability continue to be elucidated, peptides are increasingly being used to deliver either themselves or cargo to the cell’s interior.  One thing is clear, increasing the overall cationic charge of the peptide enhances it’s delivery to not only the cytoplasm, but also the nucleus or other subcellular compartments.  To achieve the positive charge, large numbers of arginine residues are most often incorporated into the peptide sequence.

This begs the question though, should I change my cleavage protocol?  In today’s post, I’ll evaluate several lengths of time used to cleave and fully deprotect an Arg-rich peptide sequence. Continue reading How long should I let my cleavage reaction stir at room temperature?

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?

Preventing aspartimide rearrangements during Fmoc-based solid phase peptide synthesis

Aspartimide rearrangements are a particularly nasty side reaction that can occur during fmoc-based solid phase peptide synthesis.  Not only is this a mass-neutral side reaction, chromatographically resolving the undesired, rearranged product can be particularly difficult.  To make matters worse, this side reaction can occur at any point during the synthesis after the Asp has been incorporated into the peptide.

In a prevous post, I described method that I have found useful for identifying whether or not an aspartimide rearrangment as occured during synthesis of a peptide that contains an aspartimide-susceptible sequence.  In today’s post, I’ll discuss some strategies that can be used to suppress, or even eliminate this side reaction. Continue reading Preventing aspartimide rearrangements during Fmoc-based solid phase peptide synthesis

Has my peptide undergone an aspartimide rearrangement?

Side reactions.  Words that cause a little shiver to run down every chemists’ spine.  As peptide chemists, we worry about both chemical side reactions like diketopiperazine or aspartimide rearrangements, and secondary structure formation as causes for failed peptide syntheses.  But how do you know what to look for?  What is a susceptible sequence and how can you confirm if one of these structural rearrangements has occurred?

In today’s post, I’ll discuss a couple strategies that have been published that illustrate how to identify if an aspartimide rearrangement has in fact occurred during your peptide synthesis. Continue reading Has my peptide undergone an aspartimide rearrangement?

Celebrating one year of the peptide synthesis and purification blog!

Wow! I can’t believe it’s been a year since this whole blogging adventure started.  This started as a technical resource for me and my colleagues within the peptide synthesis and purification space.  The idea was to enable easy access to some of the hard-to-find answers for common questions that I have encountered working with different peptide groups.

The blog has grown steadily this year and for that I thank you.  In the following post, I’ll highlight a few of the most popular posts from the past year as well a few of my favorites.  I hope you will consider providing feedback using the survey link so that we can continue to grow, keeping the content interesting and relevant.

Continue reading Celebrating one year of the peptide synthesis and purification blog!

What mobile phase flow rate should I use for my peptide purification with flash chromatography?

I’ve recently worked with several peptide groups that are trying out flash purification with their peptides for the first time.  And it never fails, every single interaction includes the question “what flow rate should I use for these cartridges?”

There is a lot of information available highlighting optimal flow rates for HPLC method development, but very little information for larger particle stationary phases.  I personally have used a wide range of flow rates for my peptide purification with differing outcomes.  So in today’s post I’d like to have a more thorough discussion about mobile phase flow rate and it’s impact on your chromatography.

Continue reading What mobile phase flow rate should I use for my peptide purification with flash chromatography?

Using mixed stationary phases to improve your peptide purification with flash chromatography

One common technique in HPLC for improving difficult peptide separations is to extend the column length, a topic I explored for flash chromatography in a previous post.  However, alternative purification strategies are sometimes necessary as the purification bottleneck grows with increasing peptide library size, both in number and scale.

In this post, I explore using two identical size cartridges in series with each packed with a different stationary phase.  I wanted to try this to see if I could improve peptide purity with the ultimate goal of reducing the time demand of peptide purification. Continue reading Using mixed stationary phases to improve your peptide purification with flash chromatography

Can I improve my peptide purification by increasing the column length?

There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC.  These strategies can include, changing column length, particle size, particle functionality (C4 vs C18).  I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.

In today’s post, I’ll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.

Continue reading Can I improve my peptide purification by increasing the column length?