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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


    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


    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


    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


    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


    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


    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


    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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