Welcome to the Biotage Peptide Synthesis Blogs.

      How to decrease your time for peptide purification optimization

      Feb 16, 2021 9:00:00 AM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Peptide Purification, Method development

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      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. 

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      Optimizing the removal of an STmp protecting group

      Dec 29, 2020 3:29:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Troubleshooting and Optimization

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      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.

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      Green solvents for solid phase peptide synthesis

      Dec 21, 2020 3:29:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Synthesis

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      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.

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

      Dec 17, 2020 3:28:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase

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      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.

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

      Dec 8, 2020 3:27:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase

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      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.

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      Optimizing a mobile phase gradient for peptide purification using flash column chromatography

      Dec 3, 2020 3:27:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Workflow

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      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.

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      Peptide Purification with flash column chromatography - a beginners experience

      Nov 24, 2020 3:26:00 PM / by Elizabeth Denton posted in Peptides, Reversed-phase

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      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.

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      Microwave heating - a route to better quality crude peptides

      Nov 19, 2020 3:26:01 PM / by Elizabeth Denton posted in microwave

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      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.

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      Getting started with Flash Chromatography for peptide purification - Tips and Tricks

      Oct 29, 2020 4:00:58 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Peptide Purification

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      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.

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      Handling difficult peptides - how to purify beta amyloid peptides

      Sep 29, 2020 3:04:09 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase

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      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.

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      How To: Measure and Optimize the Removal of MMT Protecting Groups

      Sep 22, 2020 2:41:38 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      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.

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      How to synthesize hydrophobic peptides - Choosing the Right Solvent

      Sep 1, 2020 5:39:23 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Troubleshooting and Optimization

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      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.

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      How to purify hydrophilic peptides - Why won't my peptide stick to my column?

      Jul 27, 2020 7:58:04 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Loading Techniques

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      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.

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      How to use the isoelectric point to inform your peptide purification mobile phase pH

      Jul 22, 2020 2:20:22 PM / by Elizabeth Denton posted in Reversed-phase, Alstra, Peptide Purification, Method development

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      The isoelectric point (pI) is a physical property of every peptide (and any other compound for that matter) that can be the root cause for many of the issues experienced when handling these compounds. Often times a quick check of the pI can help inform which conditions will increase or decrease aggregation potential, or lead to better solubility and generally easier handling.  

      In today's discussion, I'll demonstrate how you can use the pI to guide your purification method development for peptides.

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

      Jul 20, 2020 7:42:04 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      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.

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      Can I use a guard column for peptide purification with reversed-phase flash column chromatography?

      Jul 13, 2020 2:55:39 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Loading Techniques

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      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.  However, 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.

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