Welcome to the Biotage Peptide Synthesis Blogs.

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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.

      Read More

      How to purify synthetic peptides - what are the options?

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

      0 Comments

      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.

      Read More

      Has my peptide undergone an aspartimide rearrangement?

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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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?

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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?

      Read More

      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

      0 Comments

      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. 

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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?

      Read More

      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

      0 Comments

      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.

      Read More

      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

      0 Comments

      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.

      Read More