Welcome to the Biotage Peptide Synthesis Blogs.

      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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      How many amino acid equivalents should I use for my microwave assisted synthesis?

      Jul 13, 2020 2:53:13 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      Every now and then I work with new groups as they embark on a journey incorporating peptides and peptide synthesis into their research.  More often than not, no one in the group has experience with peptide synthesis as they are just getting operations off the ground.  As a result, one of the most common questions I receive from these groups is how much amino acid should be used during synthesis.

      In today’s post though I will address the number of equivalents of amino acid.  Large numbers of amino acid equivalents can often be used to drive coupling reactions to near completion, but the question today is how few equivalents can be used to successfully synthesize your peptide.

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      How to purify synthetic peptides - what are the options?

      Jul 7, 2020 2:15:52 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides

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      Would you ever consider an alternative to reversed- phase HPLC to purify your synthetic peptides?  It seems like a silly question, right.  And like many of you, I literally laughed at my Product Manager when he asked me this same question in my first days at Biotage.

      Fast forward a few years and my answer to that question is now very different.  For those of you that have followed this blog, you’ll know that I have switched to reversed-phase flash chromatography almost exclusively for my peptide purification.  In today’s post, I’ll highlight some of the critical reasons that have influenced my change in mindset.

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      Has my peptide undergone an aspartimide rearrangement?

      Jul 7, 2020 2:12:56 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Synthesis

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      Side reactions.  Words that cause a little shiver to run down every peptide 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.

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      How to choose your acidic mobile phase modifier for peptide purification using Reversed-Phase flash chromatography

      Jun 17, 2020 1:32:27 PM / by Elizabeth Denton posted in Troubleshooting and Optimization, Sfär, Peptide Purification

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      When purifying crude peptides, we always use a mobile phase modifier. The modifier simplifies a potentially complex purification by driving the desired peptide, and any impurities in the sample, into a single protonation state. That way, you're only trying to purify a single peptide, rather than many variants of the same peptide that only differ by the presence or absence of a couple protons - from many peaks to one!

      Acids are commonly used as mobile phase modifiers. Some groups choose trifluoracetic acid (TFA), while others choose formic acid (FA). But are there others that should be considered as well? In today's post, I'll explore the use of an acidic buffer as a mobile phase modifier and compare the purification efficiency to that observed with TFA.

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      How does mobile phase modifier concentration impact peptide purity with flash chromatography?

      Jun 2, 2020 6:44:01 PM / by Elizabeth Denton posted in Reversed-phase, Troubleshooting and Optimization, Sfär, Peptide Purification

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      We all use mobile phase modifiers, regardless of purification strategy, for purification of our synthetic peptides.  With ionizable side chains, something that affects the pH of the mobile phase is a must to ensure that the desired peptide (and any impurities) elute from the column in a single, well defined peak.  

      While a 0.1% modifier concentration is basically standard these days (sometimes you'll see different),  I started to wonder if this was really enough modifier to fully protonate all possible side chains when using flash chromatography to purify peptides.  In today's post I'll look into the effects of different mobile phase modifier concentrations with respect to general peak shape and final sample purity.

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      Peptide Workflow in action at Red Glead Discovery

      May 28, 2020 3:33:10 PM / by Amit Mehrotra posted in Peptides, Workflow, Alstra

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      Red Glead Discovery (RGD) is a pre-clinical drug discovery CRO offering a broad range of services to Life Science clients. With a focus on small molecules and peptides, their drug discovery platform ranges from medicinal chemistry and synthesis to ADME and biology. In addition to the CRO business, they also perform research collaborations with various biotech and academic partners.

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