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?

Does loading method influence my peptide recovery after purification?

In peptide purification, sample loading onto the column is rarely considered.  Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification.  If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column.  Alternatively, dry loading crude material is a strategy often used in small molecule purifications, particularly when sample solubility concerns arise.

The first question I asked myself when considering a new sample loading strategy is whether or not the purification efficiency will be maintained.  A close second though is whether or not the loading method will cause significant differences in peptide recovery.

In today’s post, I’ll compare recovery efficiencies for peptides purified using reversed phase flash chromatography but loaded onto the cartridge using either direct liquid injection or dry loaded onto reversed phase material. Continue reading Does loading method influence my peptide recovery after purification?

How much peptide is recovered from a reversed phase C18 cartridge during flash purification?

Whether it’s the bonded stationary phase, particle size, or even particle pore size, scientists today are offered a plethora of choices when it comes to reversed phase HPLC columns.  An often acknowledged concern in the peptide community though is peptide recovery from reversed phase purification efforts, particularly for precious peptide mixtures.  But how is peptide recovery impacted when you use reversed phase flash chromatography for purification?

In today’s post, I’ll compare recovery levels for two peptides that differ in length as well as crude purity using reversed phase flash chromatography.  In addition to comparing two peptides, I’ll also evaluate how recovery is impacted by altering the mobile phase pH.

Continue reading How much peptide is recovered from a reversed phase C18 cartridge during flash purification?

How many amino acid equivalents should I use for room temperature synthesis?

Big pharmaceutical companies have begun to refocus their efforts towards peptide discovery projects with the hopes of identifying the next big peptide drug.  There are often hundreds to thousands of peptides synthesized as part of these efforts, demanding parallel synthesis platforms and room temperature peptide synthesis protocols.

Previously, I identified a minimum number of amino acids equivalents required to ensure a high quality microwave synthesis.  Conducting synthesis at room temperature will certainly require different conditions than microwave heating.  Let’s explore how the number of equivalents will impact the synthesis results.

Continue reading How many amino acid equivalents should I use for room temperature synthesis?

Room temperature allyl ester and alloc deprotections – what is the lifetime of Palladium?

In a previous post, I did some work evaluating the efficiency of alloc removal with tetrakis palladium using microwave assistance and atmospheric conditions, which worked beautifully.  Given the known sensitivity of palladium catalysts (see Derek Lowe’s post for a humorous dialogue), I sought to further explore the sensitivity of palladium towards the alloc removal in the context of a peptide.

In this post, I’ll explore a variety of atmospheric, room temperature alloc deprotection conditions aimed at evaluating the catalytic lifetime of palladium tetrakis for effective alloc removal.

Continue reading Room temperature allyl ester and alloc deprotections – what is the lifetime of Palladium?

Impact of wetting an in-line guard column for reversed-phase peptide purification with flash column chromatography

More often than not and as a peptide chemist, I am asking myself in which solvent should I dissolve my peptide prior to purification by flash chromatography.  I have rarely considered an alternative to the standard liquid injection. Dry loading is a common technique used by organic chemists prior to their normal-phase purification efforts, especially if the compound isn’t particularly soluble in the mobile phase solvents.  To the best of my knowledge, dry loading is not commonly used for peptide purifications.

Immediately, questions come to mind as I attempt this new loading technique.  Will my peak shape change if load additional material?  Does the stationary phase need to be equilibrated before use?  What solvent should I use to load my crude sample? How will my sample recovery be affected?  I will address a few of these questions in today’s discussion.

Continue reading Impact of wetting an in-line guard column for reversed-phase peptide purification with flash column chromatography

Using microwave heating to expedite your allyl ester or alloc deprotection

Orthogonal amino acid protecting groups effectively expand the chemical tool kit available to peptide chemists allowing for synthesis of much more complex molecules.  Often times, orthogonal protecting groups are used in Fmoc-based chemistry to facilitate post-synthesis modifications of peptides, like the addition of small molecule fluorophores and more commonly now, peptide cyclization efforts.

In a previous post, I discussed optimizing the removal of an ivDde protecting group.  In today’s post, I’ll explore the removal of an alloc protecting group from a lysine residue.

Continue reading Using microwave heating to expedite your allyl ester or alloc deprotection