Welcome to the Biotage Peptide Synthesis Blogs.

      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 isolectric 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 isolectric 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 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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      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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      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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      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 Fundamentals, Peptides, Reversed-phase, Solvents, Detector, Isolera

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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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      Increasing your peptide purity using a focused gradient with flash chromatography

      Oct 25, 2019 10:01:02 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Sfär, Selekt

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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 does media pore size impact peptide resolution?

      Sep 27, 2019 3:19:01 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Green, Sfär, Media and Resin, Cost, V-10, HPLC

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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 Fundamentals, Peptides, Reversed-phase, HPLC

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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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      How to purify your peptide using mass directed flash chromatography

      Jul 31, 2019 4:02:19 PM / by Elizabeth Denton posted in Peptides, Reversed-phase, Selekt, Detector, Isolera

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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 Fundamentals, Peptides, Reversed-phase, Sfär

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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 Fundamentals, Peptides, Reversed-phase, Solvents, Selekt, Isolera

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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 Peptides, Reversed-phase, Sfär, HPLC

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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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      How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?

      Jul 18, 2019 2:10:15 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Solvents, Green, Sfär

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      Recently there has been substantial motivation to consider and evaluate alternative, more environmentally friendly solvents.  Some countries have even gone so far as to ban some of the more toxic, yet commonly used solvents.  In addition to general toxicity, additional consideration in the green chemistry movement is the volume of solvent used in any particular application.  In this regard, purification solvent selection is closely monitored as they are often used in large quantities.

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      Does loading method influence my peptide recovery after purification?

      Jul 17, 2019 6:25:29 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase, Solvents, Workflow, Sfär, Loading Techniques, V-10, Isolera

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      In peptide purification, sample loading onto the column is rarely considered.  Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification.  If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column.  Alternatively, dry loading crude material is a strategy often used in small molecule purification, particularly when sample solubility concerns arise.

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      Can I improve my peptide purification by increasing the column length?

      May 23, 2019 4:41:39 PM / by Elizabeth Denton posted in Chromatography Fundamentals, Peptides, Reversed-phase

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      There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC.  These strategies can include, changing column length, particle size, particle functionality (C4 vs C18).  I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.

      In today's post, I'll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.

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