Welcome to the Biotage Peptide Synthesis Blogs.

      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

      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, Peptides, Reversed-phase, Solvents

      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

      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

      0 Comments

      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.

      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 Optimization, Peptides, Reversed-phase, Solvents

      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, Peptides, Reversed-phase, Loading method

      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

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

      Jun 19, 2019 5:35:32 PM / by Elizabeth Denton posted in Peptides, Workflow

      0 Comments

      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.

      Read More

      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 Peptides, Synthesis

      0 Comments

      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. 

      Read More

      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, Synthesis

      0 Comments

      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.

      Read More

      Can you use normal phase chromatography to purify protected peptides?

      Jun 11, 2019 8:29:38 PM / by Elizabeth Denton posted in Chromatography, Normal phase, Peptides, Reversed-phase

      0 Comments

      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.

      Read More

      Using microwave heating to expedite your allyl ester or alloc deprotection

      Jun 11, 2019 8:28:51 PM / by Elizabeth Denton posted in Peptides, Synthesis

      0 Comments

      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.

      Read More

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

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

      0 Comments

      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.

      Read More

      Using double coupling to improve your peptide synthesis

      Apr 29, 2019 6:06:48 PM / by Elizabeth Denton posted in Peptides, Synthesis

      0 Comments

      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.

      Read More

      Optimizing the removal of an ivDde protecting group

      Apr 29, 2019 6:05:57 PM / by Elizabeth Denton posted in Peptides, Synthesis

      0 Comments

      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.

      Read More

      How To Load The First Amino Acid Onto Wang Resin

      Apr 29, 2019 6:05:23 PM / by Elizabeth Denton posted in Peptides, Synthesis, Resin

      0 Comments

      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.

      Read More

      How to quantify your first amino acid loading onto Wang resins

      Apr 29, 2019 6:04:33 PM / by Elizabeth Denton posted in Peptides, Synthesis, Resin

      0 Comments

      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.

      Read More

      How long are amino acid stock solution stable for successful solid phase peptide synthesis?

      Apr 29, 2019 6:03:52 PM / by Elizabeth Denton posted in Peptides, Synthesis

      0 Comments

      In today’s post I’ll answer the above question by comparing the crude purity of peptides synthesized using amino acid stock solutions or freshly dissolved amino acids.

      Read More