Breakthrough during reversed phase purification often occurs for only a couple of reasons: 1) the dissolution solvent is too strong and prevents the compound from fully interacting with the stationary phase or 2) too much sample has been loaded onto the column. Peptides, particularly when part of a crude sample mixture, are known for their minimal solubility in aqueous conditions which further complicates purification efforts and often compromises recovery of purified material.  This problem is further is exacerbated when the peptide of interest is small and contains polar amino acid(s), which often results in poor retention by the C18 stationary phase commonly used in peptide purification.

In today's discussion, I'll highlight an unusual strategy to address both solubility and retention, while decreasing the breakthrough that can occur during purification of these types of peptides.

Synthesizing libraries of peptides - potentially hundreds in a single synthesizer setup - has seen a resurgence of late.  These synthetic libraries, often prepared on very small synthesis scales, are required most recently for secondary screening preliminary structure-activity relationship determinations, or neoantigen vaccine preparations.  However, optimizing the synthesis of hundreds of peptides to be made simultaneously is a very different beast when compared to optimizing the synthesis of a single peptide.

In today's post, I'll describe a strategy that can increase the overall crude purity of an entire peptide library using two different coupling chemistries.

We’ve all done it. Loaded a very small amount of our peptide onto an analytical, maybe semi-prep column, to see where the desired peak elutes, then create a shortened version of that gradient for the actual purification runs. A scouting run like the above mentioned gives the chemist a lot of information about the sample – resolution from impurities, potential gradient slopes to improve the separation, even loading capacity can be calculated.

But how many times have you used a scouting run like this to figure out the purification gradient, only to have everything change when you increased your sample load? In today’s post, I highlight how easy scaling - aka increasing your sample load - can be accomplished when using reversed phase flash chromatography for peptide purification and will include some tips for success along the way.

Let's be honest.  Optimizing a purification protocol for a single peptide is not something that one wants to spend a lot of time thinking about, let alone actually pursuing.  There are times though, that a little bit of effort in this arena can very impactful when considering the time required to fully synthesize, purify and deliver a peptide. 

In today's post, I'll discuss an approach combining the use of a focused gradient with a step gradient that minimizes the steps necessary to identify a more optimal flash purification gradient for peptide purification. 

Disulfide rich peptides are unique in both their incredibly high cysteine content, but also in the stability imbued by the multiple disulfide bonds.  These peptides, stable under extreme conditions that would either denature or degrade a similar linear peptide, make disulfide rich peptides attractive as both therapeutics or as scaffolds upon which to construct non-native functionality.  Synthesizing these compounds, however, still remains a challenge.

I have discussed previously strategies that enable on-resin chemistry via orthogonal protecting groups.  These groups can be removed under mildly acidic, metal catalyzed, or even oxidizing conditions.  In today’s post, I’ll demonstrate the utility of using disulfide shuffling as a cysteine protection strategy.

As peptide therapeutics continue to gain interest from the medical community and pharmaceutical companies, concerns regarding the cost of manufacturing also grow.  Cost  includes the expense of reagents and solvents, including DMF and NMP, used in the synthesis but also subsequent disposal.  

With growing motivations to improve the "green" characteristics of chemistry (reducing waste, reducing use of harmful solvents, etc), today's post will highlight some recent work evaluating alternative solvents for use in solid phase peptide synthesis.

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.

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.

Have you ever wondered if there was a faster (and possibly 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.

As a peptide chemist, I was trained to purify my peptides with reversed-phase HPLC, just as many a peptide chemist before me. Despite the hundreds of hours I’ve logged in front of an HPLC, injecting samples and collecting peak fractions, I can’t imagine using any other method to purify my freshly synthesized and cleaved peptides.  In fact, you’d be hard pressed to convince me to try something else.  But here I am, trying something new.  Wish me luck!

In this post, I’ll describe my first experiences using flash chromatography to purify a peptide sample.

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.

Have you ever tried a new-to-you technique, only to find yourself frustrated by the lack of results?  Frustration that only seems to compound when you search for resources that may help you get started and only to come up empty-handed?  Me too!!  And often for me, this effort ends with me back to using whatever the previous strategy may have been, because whether it's efficient or not, it's the devil I already know.  

I've been working with flash chromatography as my primary tool for peptide purification for a couple of years now and honestly encountered some of these similar experiences.  But rather than give up, I pushed on and with the help of some other scientists, have identified some strategies that can help make this technique much easier.

In today's post, I've compiled a few of the most helpful strategies into a tips and tricks list that should help you to be more successful using flash chromatography for your peptide purification.

Sometimes the most interesting peptides are also the absolute worst to work with.  Whether it's synthetic difficulty, or solubility issues, or purification difficulty, or worst of them - all of the above - the experiments must go on!  This is the case for amyloid beta and many of the amyloidogenic peptides currently being studied.  

In today's post, I'll use this particularly difficult peptide to handle as a case study for some strategies to improve your purification efficiency.

Orthogonal side chain protecting groups, particularly for Fmoc-based solid phase peptide synthesis, are growing not only in diversity, but also in popularity.  These protecting groups enable post-synthesis chemistry while the peptide is still on resin, often times increasing efficiency, decreasing side reactions, and generally simplifying the overall process.

I've already done some work with many of the commercially available orthogonally protected amino acids including allyl and alloc, Acm, and ivDde for a variety of downstream applications.  In today's post, I'll discuss some work optimizing the removal of a 4-methoxytrityl (Mmt) group from cysteine side chains.

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.

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.