Welcome to the Biotage Peptide Synthesis Blogs.

      How to choose your acidic mobile phase modifier for peptide purification using Reversed-Phase flash chromatography

      Jun 17, 2020 1:32:27 PM / by Elizabeth Denton posted in Troubleshooting and Optimization, Sfär, Peptide Purification

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      When purifying crude peptides, we always use a mobile phase modifier. The modifier simplifies a potentially complex purification by driving the desired peptide, and any impurities in the sample, into a single protonation state. That way, you're only trying to purify a single peptide, rather than many variants of the same peptide that only differ by the presence or absence of a couple protons - from many peaks to one!

      Acids are commonly used as mobile phase modifiers. Some groups choose trifluoracetic acid (TFA), while others choose formic acid (FA). But are there others that should be considered as well? In today's post, I'll explore the use of an acidic buffer as a mobile phase modifier and compare the purification efficiency to that observed with TFA.

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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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      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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      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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      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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      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 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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      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 you use normal phase chromatography to purify protected peptides?

      Jun 11, 2019 8:29:38 PM / by Elizabeth Denton posted in Peptides, Solvents, Sfär, Normal Phase, Isolera

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      Chemical synthesis of peptides, and even proteins, offers the possibility to expand the functionality and stability imbued by nature.  However, chemical synthesis of very long peptides and small proteins remains today an exceedingly difficult task.  Several ligation strategies have been developed that help to alleviate this challenge.  These strategies though, require a purified, yet fully protected peptide fragment.

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      How St. John's University Accelerated their Cancer Research with the Biotage Peptide Workflow Solution

      Jan 11, 2019 4:14:31 PM / by Sarah Moran posted in Peptides, Synthesis, Workflow, Sfär, Customer case, Alstra, V-10, Isolera

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      "How can I take a two hour purification using old-school chromatography, and shorten it?" was the question Dr. Aaron Muth at St. John's University in New York asked himself. "By using the Biotage Isolera, I could take it down to 10 or 15 minutes."

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