Welcome to the Biotage Peptide Synthesis Blogs.

      Optimizing the removal of an ACM protecting group

      Nov 14, 2019, 3:40:28 PM / by Elizabeth Denton posted in Optimization, Peptides, Troubleshooting, Synthesis

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      Disulfide rich peptides have gained significant attention recently due to their incredible biological stability and tolerance to epitope grafting.  This class of peptides is often folded in solution, assuming the desired disulfide bond pattern correlates with the most thermodynamically stable structure.  Sometimes though, especially for chemically synthesized cysteine rich peptides, this is not the case.  The result is a complex mixture of peptides with varying disulfide bonding patterns and identical mass.

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      How do I choose my resin for peptide synthesis? {Part 2}

      Nov 8, 2019, 4:23:13 PM / by Elizabeth Denton posted in Optimization, Peptides, Synthesis

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      Resins for solid phase peptide synthesis can vary significantly in both functionalization and composition, leading to mixed results at the end of a synthesis.  Previously, I demonstrated how the resin loading level affects the success or failure of your peptide synthesis.

      In today’s post, I’ll highlight how both the hydrophilicity and swelling capacity of your resin can influence your peptide synthesis.

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      How does my mass spectrometer carrier solvent impact mass-directed purification of peptides?

      Nov 8, 2019, 4:18:50 PM / by Elizabeth Denton posted in Chromatography, Peptides, Reversed-phase, Solvents, Mass-directed purification

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      Mass-directed purification, whether with a preparative HPLC or a bench-top flash system, is quickly gaining interest in the peptide purification space.  The simple fact is that using a specific mass, rather that UV absorbance, to trigger fraction collection allows for greater confidence in the identity of the collected fraction.  Importantly though, this technique can also reduce your time required for purification, by significantly reducing or even eliminating the need for secondary mass analysis of each collected fraction. 

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      Does amino acid concentration really matter during peptide synthesis?

      Nov 8, 2019, 4:15:44 PM / by Elizabeth Denton posted in Optimization, Peptides, Synthesis

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      When it comes to synthesizing a peptide, the first thing that comes to mind is the number of stoichiometric equivalents to use.  Sometimes that number is as few as 1.5, sometimes it’s as high as 20!

      But have you ever thought about the liquid volume that contains those molecules and how that might affect the success of your coupling reaction?  In this post I will discuss the impact of amino acid concentration in the overall success of solid phase peptide synthesis.

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      How do I choose my resin for peptide synthesis? {Part 1}

      Nov 8, 2019, 4:13:33 PM / by Elizabeth Denton posted in Optimization, Peptides, Synthesis

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      It used to be easy with only polystyrene based resin types, but nowadays there is a broad choice of types to choose from, including everything from the C-terminal functionality (Rink vs Wang) to the polymer from which the resin itself is synthesized.

      All resins have one thing in common, and that’s the reactive site loading level. In this post, I will share my experiences with how this important factor impacts the success of peptide synthesis.

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      Increasing your peptide purity using a focused gradient with flash chromatography

      Oct 25, 2019, 10:01:02 PM / by Elizabeth Denton posted in Chromatography, Optimization, Peptides

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      Reversed phase flash chromatography is increasingly being utilized by peptide chemists to decrease purification time and efforts. The larger particles used in flash columns enable large crude sample loads and can lead to highly pure peptide samples despite lower resolution when compared to traditional HPLC methods. However, there are some situations where the purity achieved isn't sufficient. Then what can you do?

      In today's post, I'll describe using a focused gradient to achieve higher purity peptides than is possible with a more traditional linear gradient.

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      How long should I let my cleavage reaction stir at room temperature?

      Sep 27, 2019, 3:30:52 PM / by Elizabeth Denton posted in Optimization, Peptides, Mass-directed purification

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      As the rules for cell permeability continue to be elucidated, peptides are increasingly being used to deliver either themselves or cargo to the cell’s interior.  One thing is clear, increasing the overall cationic charge of the peptide enhances it’s delivery to not only the cytoplasm, but also the nucleus or other subcellular compartments.  To achieve the positive charge, large numbers of arginine residues are most often incorporated into the peptide sequence.

      This begs the question though, should I change my cleavage protocol?  In today’s post, I’ll evaluate several lengths of time used to cleave and fully deprotect an Arg-rich peptide sequence.

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      Five Tips and Tricks for Success in Solid Phase Peptide Synthesis

      Sep 27, 2019, 3:26:17 PM / by Elizabeth Denton posted in Optimization, Peptides

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      In my role as a peptide application scientist, I have had the pleasure of working with many groups that are venturing into the world of peptides for the first time.  Although it seems rather  straightforward to experienced synthetic chemists, producing acceptable yield and purity certainly comes with unique challenges in solid phase peptide synthesis .

      In this post I would like to present some of the tips and tricks that I have picked up along the way.

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      How does media pore size impact peptide resolution?

      Sep 27, 2019, 3:19:01 PM / by Elizabeth Denton posted in Chromatography, Peptides, Reversed-phase, Media

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      Purification by reversed-phase chromatography relies primarily on a hydrophobic interaction of the molecule with the alkyl chains bonded to the stationary phase for column retention and elution through a partitioning mechanism.  While this is certainly true for purification of peptides, surface area accessibility and media particle size also play critical roles in the resolving power of a particular stationary phase.  The particle size influences the loading capacity, however pore size greatly influences molecular accessibility and therefore resolving power.

      In today’s post, I will demonstrate how pore size can impact your peptide purification using flash column chromatography.

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      How to use a scouting column for your peptide purification

      Sep 27, 2019, 3:13:39 PM / by Elizabeth Denton posted in Chromatography, Optimization, Peptides, Reversed-phase, Media

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      In the past, when I have synthesized a new peptide, I always ran a “scout run” – a small scale injection, usually with an analytical HPLC column – to both get an idea of the crude purity and also to identify a shorter, more optimal gradient for the actual purification.  This strategy is still recommended when you want to use reversed phase flash chromatography for your purification strategy, but is there a better way?

      In today’s post, I’ll discuss using a scouting column to screen gradient conditions prior to peptide purification with reversed phase flash chromatography.

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      What is solid phase peptide synthesis?

      Sep 25, 2019, 9:47:21 PM / by Elizabeth Denton posted in Optimization, Peptides, Synthesis

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      More and more groups are exploring the utility of peptides with an ever widening variety of applications. And although peptides are getting cheaper to purchase outright, many groups are continuing to bring peptide synthesis in house. As more groups join the peptide community, I frequently encounter questions about the basics of peptide synthesis.

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      Peptides containing cysteine: the role of scavengers in cleavage cocktail

      Sep 11, 2019, 10:48:03 PM / by Elizabeth Denton posted in Peptides

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      Since the development of Fmoc-based solid phase peptide synthesis, a wide variety of cleavage cocktails have emerged.  Each cleavage cocktail contains a unique combination of scavengers designed to prevent either side reactions mediated by the released protecting groups or the side chains themselves, or both during the peptide cleavage reaction.  As the number of scientists performing peptide synthesis grows, the question “which cleavage cocktail should I use?” comes up more often than not.

      In today’s post, I’ll highlight the role of of scavengers for peptides containing cysteine residues.

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      How to purify your peptide using mass directed flash chromatography

      Jul 31, 2019, 4:02:19 PM / by Elizabeth Denton posted in Chromatography, Optimization, Peptides, Media, Mass-directed purification

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      We've all used mass spectrometry to characterize our synthetic peptides.  It's often used to confirm that the peptide was in fact synthesized, then again as part of the purification process to make sure that we're collecting the correct peak.  But how many of you had the opportunity to use in-line mass spectrometry as an integral component during the purification itself?

      In today's post, I'll highlight some of the advantages to using in-line mass mass spectrometry for purification of peptides using reversed phase flash chromatography.

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      How much peptide is recovered from a reversed-phase C18 cartridge during flash purification?

      Jul 25, 2019, 2:13:43 PM / by Elizabeth Denton posted in Chromatography, Peptides, Reversed-phase

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      Whether it’s the bonded stationary phase, particle size, or even particle pore size, scientists today are offered a plethora of choices when it comes to reversed phase HPLC columns.  An often acknowledged concern in the peptide community though is peptide recovery from reversed phase purification efforts, particularly for precious peptide mixtures.  But how is peptide recovery impacted when you use reversed phase flash chromatography for purification?

      In today’s post, I’ll compare recovery levels for two peptides that differ in length as well as crude purity using reversed phase flash chromatography.  In addition to comparing two peptides, I’ll also evaluate how recovery is impacted by altering the mobile phase pH.

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      How to choose an ion pairing agent to improve your peptide purification

      Jul 22, 2019, 4:28:04 PM / by Elizabeth Denton posted in Chromatography, Peptides, Reversed-phase, Solvents

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      Ion pairing agents are used in a variety of strategies to improve overall purification efficiency. In a previous post, I utilized ion pairing agents to increase the peptide’s hydrophobicity, improving retention by the stationary phase and enabling purification.  But what other strategies can be improved by using ion pairing agents?

      In this post, I’ll utilize ion pairing agents to enable rapid peptide purification by flash chromatography.  The use of ion pairing agents can in fact alter the peptide’s apparent hydrophobicity sufficiently that the desired peptide and it’s closely eluting impurities can be resolved.  The question is, which one to choose?

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      Peptide purification with flash column chromatography - a beginner's experience

      Jul 18, 2019, 8:59:34 PM / by Elizabeth Denton posted in Chromatography, 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 experiences using flash chromatography to purify a new peptide sample.

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