Welcome to the Biotage Peptide Synthesis Blogs.

      How to improve peptide purification by altering the mobile phase pH

      Apr 17, 2020 11:45:00 AM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Solvents

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      Peptides, by nature, are composed of amino acids with potentially ionizable chemical moieties. The ionization state of any of these moieties can significantly impact the peptide’s chromatographic behavior, both in terms of peak shape and retention by the solid support.  Peptide purification by reversed-phase chromatography, however, almost exclusively includes an acidic additive to the mobile phase solvents, maintaining the solution at a pH of 2-3 throughout the purification cycle.  But have you ever considered trying an alternative additive in the mobile phase to improve your purification results?

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      Using Mixed Stationary Phases to Improve Your Peptide Purification with Flash Chromatography

      Apr 15, 2020 6:00:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Sfär

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      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.

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      Cyclic Peptides Webinar Recap

      Apr 15, 2020 2:40:31 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Webinar, Automation, Alstra

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      Peptides, while exhibiting potential for unique specificity and affinity, still suffer from stability issues when introduced to a biological system.  One strategy to overcome these stability issues is include a covalent bond, creating a peptide macrocycle.  

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      Disulfide Rich Peptides - which order should the disulfide bonds be formed?

      Apr 15, 2020 2:38:56 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Synthesis, Alstra

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      Disulfide rich peptides are being identified in species of both plants and animals at increasing rates. As new molecules are discovered and disulfide bonding patterns characterized, the need for simplified chemical synthesis strategies is also increasing.

      I have previously written about optimizing removal of several orthogonal side chain protecting groups including allyl, alloc, ivDde and acetamidomethyl (Acm) groups. The question that I’ll address today, though, is does the order in which the disulfide bonds are formed matter for cleaning up reactions to produce chemically synthesized disulfide rich peptides?

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      Which Stationary Phase Should I Chose For My Peptide Purification?

      Apr 12, 2020 6:45:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Sfär, Media and Resin

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      Almost all the peptides I have synthesized were subsequently purified using a reversed-phase C18 column.  Sometimes this worked, but sometimes it didn’t work so well.  When my C18 purifications failed, I questioned whether or not I could have predicted this outcome prior to extensive HPLC efforts.  Since then, I have learned that the amino acid composition of the peptide may give some clues to the peptide’s chromatographic behavior.

      While there are numerous stationary phase functionalization types for reversed-phase chromatography, in today’s post I will describe some differences I have observed when purifying peptides using C18- or C4- functionalized stationary phases for peptide purification.

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      Preventing Aspartimide Rearrangements During Fmoc-based Solid Phase Peptide Synthesis

      Apr 9, 2020 1:15:00 PM / by Elizabeth Denton posted in Peptides, Troubleshooting and Optimization, Synthesis

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      Aspartimide rearrangements are a particularly nasty side reaction that can occur during fmoc-based solid phase peptide synthesis.  Not only is this a mass-neutral side reaction, chromatographically resolving the undesired, rearranged product can be particularly difficult.  To make matters worse, this side reaction can occur at any point during the synthesis after the Asp has been incorporated into the peptide.

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      How does flow rate affect my peptide purification efficiency when using a small pore stationary phase

      Apr 6, 2020 6:08:00 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Troubleshooting and Optimization, Sfär, Selekt, Media and Resin

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      In a previous post, I evaluated how flow rate can impact my purification efficiency using flash chromatography.  I noticed though, that my peptide eluted significantly later with high mobile phase flow rates.  I hypothesized that the increased pressure (caused by higher flow rates) was driving the compound further into the pores, increasing the overall interaction with the stationary phase and causing the increased retention.  We know that the particle size and particle pore size impact resolution and purification efficiency, so how does flow rate play a role with a different stationary phase?

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      Automating stapled peptide synthesis: overcoming DMF poisoning

      Apr 1, 2020 9:16:05 PM / by Elizabeth Denton posted in Troubleshooting and Optimization

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      Covalent stapling strategies that stabilize a particular secondary structure have garnered much attention as interest in peptide therapeutics continues to grow.  One such strategy - using olefin-bearing unnatural amino acids covalently bonded using ring-closing metathesis - has been exploited to the greatest extent thusfar.

      In today's post, I'll discuss some strategies to overcome DMF poisoning of the Grubbs catalyst used during the metathesis reaction towards fully automating the synthesis and secondary chemistry required for stapled peptides.

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