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      Elizabeth Denton


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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 Developments, Mass Detection, solid phase peptide synthesis, purify, loading capacity, peptide purification, peptides and flash chromatography, 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 Developments, reversed-phase, flash chromatography, peptide, solid phase peptide synthesis, peptide purification, peptides and flash chromatography

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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 Developments, reversed-phase, flash chromatography, peptide, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, ion pairing agents

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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 Developments, Peptides, reversed-phase, flash chromatography, solid phase peptide synthesis, peptide purification

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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 Peptides, reversed-phase, selectivity, solvent strength, solid phase peptide synthesis, peptide purification

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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.

      One alternative that is growing in popularity is the use of methanol in place of acetonitrile for reversed phase purification of small molecules.  Methanol is certainly less expensive, but is also a more environmentally-friendly solvent for use in purification applications.  But it’s use for peptide purification has not been widely adopted to date.  In today’s post, I’ll compare the purification efficiency of methanol when compared to acetonitrile for peptide purification by reversed phase flash chromatography.

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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 Peptides, reversed-phase, solid phase peptide synthesis, peptide purification, peptides and flash chromatography, loading method

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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.

      The first question I asked myself when considering a new sample loading strategy is whether or not the purification efficiency will be maintained.  A close second though is whether or not the loading method will cause significant differences in peptide recovery.

      In today's post, I'll compare recovery efficiencies for peptides purified using reversed phase flash chromatography but loaded onto the cartridge using either direct liquid injection or dry loaded onto reversed phase material.

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      Post synthesis workup: What steps are necessary and what aren't?

      Jun 19, 2019 5:35:32 PM / by Elizabeth Denton posted in Developments, Peptides, workflow, peptide workflow, solid phase peptide synthesis, cleavage

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      You’ve just finished a peptide synthesis and now it’s time to cleave the peptide from the resin. You’ve selected a specific cleavage cocktail, performed the reaction and now what? The vast majority of peptide chemists will precipitate their peptide using an ether solution, lyophilize, and move on to purification. But is that the only option?

      In today’s post I’ll highlight an alternative strategy that saves both processing time, potentially dangerous reagents, all without compromising the integrity of the recently synthesized peptide.

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      Room temperature allyl ester and alloc deprotections - what is the lifetime of palladium?

      Jun 11, 2019 8:30:33 PM / by Elizabeth Denton posted in solid phase peptide synthesis, orthogonal protecting groups, selecting deprotection, synthesis optimization

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      In a previous post, I did some work evaluating the efficiency of alloc removal with tetrakis palladium using microwave assistance and atmospheric conditions, which worked beautifully.  Given the known sensitivity of palladium catalysts (see Derek Lowe's post for a humorous dialogue), I sought to further explore the sensitivity of palladium towards the alloc removal in the context of a peptide.

      In this post, I'll explore a variety of atmospheric, room temperature alloc deprotection conditions aimed at evaluating the catalytic lifetime of palladium tetrakis for effective alloc removal. 

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      How to choose the right resin functionality for solid phase peptide synthesis

      Jun 11, 2019 8:30:01 PM / by Elizabeth Denton posted in Peptides, solid phase peptide synthesis, synthesis tips, synthesis optimization

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      As a chemist new to the peptide community, there are many choices that have to be made.  Which coupling reagents to use? Heat or no heat to promote chemistry? And most importantly, which resin?  I have talked previously about resin choices, from loading levels to swelling capacity and how they affect the synthesis outcome.  But I haven't addressed yet a fundamental feature of commercially available resins, and that's the functional handle to which the peptide chain is conjugated.

      In today's post, I'll describe some, and I mean only some, of the most commonly used chemical functionalities for Fmoc-based solid phase peptide synthesis and some scenarios in which you would choose one resin type over another.

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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 normal phase, reversed-phase, flash purification, peptide synthesis, solid phase peptide synthesis, peptides and flash chromatography

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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.

      Purification of a fully protected peptide species can be challenging by standard reversed-phase techniques, primarily due to the limited solubility of protected peptides in aqueous solutions.  In today’s post, I will discuss using normal-phase chromatography for purification of protected peptides.

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      Using microwave heating to expedite your allyl ester or alloc deprotection

      Jun 11, 2019 8:28:51 PM / by Elizabeth Denton posted in peptide, peptide synthesis, solid phase peptide synthesis, orthogonal protecting groups, synthesis optimization

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      Orthogonal amino acid protecting groups effectively expand the chemical tool kit available to peptide chemists allowing for synthesis of much more complex molecules.  Often times, orthogonal protecting groups are used in Fmoc-based chemistry to facilitate post-synthesis modifications of peptides, like the addition of small molecule fluorophores and more commonly now, peptide cyclization efforts.

      In a previous post, I discussed optimizing the removal of an ivDde protecting group.  In today’s post, I’ll explore the removal of an alloc protecting group from a lysine residue.

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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 reversed-phase, peptide workflow, v-10 touch, solid phase peptide synthesis, peptide purification, peptides and flash chromatography

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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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      Using double coupling to improve your peptide synthesis

      Apr 29, 2019 6:06:48 PM / by Elizabeth Denton posted in peptide workflow, solid phase peptide synthesis, synthesis tips

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      There are several strategies employed when a peptide synthesis requires optimization.  Typically, the first thing considered is whether or not to double couple specific amino acids within the sequence.  This is somewhat of a change in mentality from traditional room temperature synthesis strategies where double coupling is frequently used for the entire peptide sequence.

      In a previous post, I briefly described several scenarios in which doubling coupling can be used in conjunction with microwave heating to improve the overall crude peptide purity.  In today’s post, I will delve more deeply into the question of whether or not double coupling is necessary to improve your peptide synthesis.

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      Optimizing the removal of an ivDde protecting group

      Apr 29, 2019 6:05:57 PM / by Elizabeth Denton posted in peptide workflow, solid phase peptide synthesis, synthesis tips

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      As the complexity of peptides continues to grow, so does the use of amino acids with side chain protecting groups that can be selectively removed, leaving the peptide on resin and the remaining side chain protecting groups intact.  While there are  protocols to be found in the literature, they may not work to the highest level of efficiency every single time.  This can lead to disasterous results for any subsequent chemistry.

      In today’s post, I’ll evaluate a variety of conditions for removing an ivDde protecting group from the lysine side chain amine.

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      How To Load The First Amino Acid Onto Wang Resin

      Apr 29, 2019 6:05:23 PM / by Elizabeth Denton posted in peptide workflow, solid phase peptide synthesis, first amino acid loading, resin loading, synthesis tips

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      While resins loaded with the natural 20 amino acids are commercially available these days, there may be times when loading the first amino acid onto the resin in house may be necessary.  And unlike loading the first amino acid onto amide-leaving resins, the first coupling reaction for C-terminal acids can be chemically more challenging.

      There are several protocols published both in the literature as well as in technical notes from many peptide reagent and instrument suppliers, but they typically occur at room temperature over extended periods of time (3-24 hours and repeated).  In today’s post, I’ll evaluate several conditions suitable for efficiently loading the first amino acid onto Wang-type resin.

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      How to quantify your first amino acid loading onto Wang resins

      Apr 29, 2019 6:04:33 PM / by Elizabeth Denton posted in solid phase peptide synthesis, quantifying resin loading, first amino acid loading, resin loading, synthesis tips

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      While many of the standard amino acids can be purchased pre-loaded onto Wang type resins, there are still cases where coupling the first amino acid onto Wang resin manually is necessary.  In my case, an unnatural amino acid was required on the C-terminus so there was not a commercially available source.
      This coupling reaction comes with it’s own set of challenges, which is why many people perform a large scale batch preparation of the pre-loaded resin.  But that’s for a later discussion.  In today’s post I’ll address a different question. How do you quantify the amount of amino acid loaded onto the resin?

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