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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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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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 with flash column chromatography – a beginner’s experience

With this post I would like to introduce a new colleague, Beth Denton, who I recently convinced to try flash chromatography for purification of peptides. She has kindly agreed to share her new flash column chromatography experience here.  So, now, here’s Beth.

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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