Using double coupling to improve your peptide synthesis

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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How do I choose my resin for peptide synthesis? Part 2

Resins for solid phase peptide synthesis can vary significantly in both functionalization and composition, leading to mixed results at the end of a synthesis.  Previously, I demonstrated how the resin loading level affects the success or failure of your peptide synthesis.

In today’s post, I’ll highlight how both the hydrophilicity and swelling capacity of your resin can influence your peptide synthesis.

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How do I choose my resin for peptide synthesis? Part 1

It used to be easy with only polystyrene based types, but nowadays there is a broad choice of types to choose from, including everything from the C-terminal functionality (Rink vs Wang) to the polymer from which the resin itself is synthesized.

All resins have one thing in common, and that’s the reactive site loading level. In this post, I will share my experiences with how this important factor impacts the success of peptide synthesis.

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Peptide purification with flash column chromatography – a beginner’s experience

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 media pore size impact peptide resolving power?

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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Optimizing a mobile phase gradient for peptide purification using flash column chromatography

Have you ever wondered if there was a faster and cheaper way to purify your peptides?

My colleagues and I in the peptide community rely almost exclusively on reversed-phase HPLC for delivery of highly pure peptide products.  However, this process is often very time consuming and requires expensive columns and solvents to be successful.  Alternatively, peptide purification via reversed-phase flash column chromatography can be used to complete a purification in a fraction of the time and with a fraction of the costs.

Here I will show how I do gradient optimization for peptide purification via reversed-phase flash column chromatography and will highlight the similarities with standard HPLC methodologies.

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Peptide purification improvements with flash column chromatography by modulating mobile phase pH

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?

In the following post I discuss the impact of mobile phase pH in the purification of oxytocin (CYIQNCPLG-NH2), a 9-amino acid peptide that requires disulfide bond-mediated cyclization for its biological activity.

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Beth’s top Five Tips and Tricks for Success in Solid Phase Peptide Synthesis

In my role as a peptide application scientist, I have had the pleasure of working with many groups that are venturing into the world of peptides for the first time.  Although it seems rather  straightforward to experienced synthetic chemists, producing acceptable yield and purity certainly comes with unique challenges in solid phase peptide synthesis .

In this post I would like to present some of the tips and tricks that I have picked up along the way.

Find them out here!

Does amino acid concentration really matter during peptide synthesis?

When it comes to synthesizing a peptide, the first thing that comes to mind is the number of stoichiometric equivalents to use.  Sometimes that number is as few as 1.5, sometimes it’s as high as 20!

But have you ever thought about the liquid volume that contain those molecules and how that might affect the success of your coupling reaction?  In this post I will discuss the impact of amino acid concentration in the overall success of solid phase peptide synthesis.

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