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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Can reversed-phase flash column chromatography be greener?

In previous posts I offered some suggestions to improve the “greenness” of normal-phase flash purification.  As the use of reversed-phase flash purification has increased the past few years I thought I would explore how to potentially make it greener too.

So, with that in mind, let’s take a look at the use of acetone in place of acetonitrile as a reversed-phase flash chromatography solvent. Continue reading Can reversed-phase flash column chromatography be greener?

Purifying ionic compounds by flash column chromatography

One of the more challenging purifications is that of water-soluble, ionizable compounds. Typically, normal-phase with silica is not used because of the probable non-reversible interactions, especially between the ionized amines interacting and the ionizable silanols.  With normal-phase out of the purification solution that leaves ion exchange and reversed-phase as chromatographic options.

In this post I will discuss the use of reversed-phase and the influence pH and buffers have on the chromatography of some ionic, water soluble compounds. Continue reading Purifying ionic compounds by flash column chromatography

How do I determine loading capacity in reverse phase flash column chromatography?

As the popularity of prep-scale, reversed-phase flash chromatography increases, so does the frequency that I get asked this question, “How do I determine loading capacity in reversed-phase flash chromatography?”

In the world of HPLC, loading capacity isn’t normally a concern as it is primarily an analytical technique.  In the synthetic organic chemistry world, most purification is performed with silica gel where flash column purification methods are developed and loading capacity estimated from TLC data.  However, when normal-phase flash does not work and reversed-phase flash is needed, the question of how to determine reversed-phase loading capacity comes up.

In this post I will attempt to provide some guidelines to help you understand and determine reversed-phase flash chromatography loading capacity. Continue reading How do I determine loading capacity in reverse phase flash column chromatography?

Cannabis extract purification using orthogonal flash column chromatography

For some cannabis-based product developers reversed-phase chromatography has become the analytical tool of choice for determining the extract content profile as well as for purification of specific cannabinoid compounds. However, the extracts often contain many other compounds which reduce load capacity and purity of the product(s) of interest and then require even more extensive clean-up.

In this post I show the results of an orthogonal flash purification approach that first uses normal-phase flash column chromatography to clean up the crude cannabis extract followed by reversed-phase C18 flash chromatography of the isolated target compounds.  This orthogonal approach to purification increases the targeted product’s purity.

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Detecting the undetectable in flash column chromatography, part 2

Compound detection challenges are, for many chemists, a part of life. In a previous post I discussed how wavelength focusing can help your flash system detect and fractionate compounds with poor chromophores. However, compounds naked to UV-Vis light,  such as carbohydrates, are impossible to detect by UV when separating by liquid chromatography.

There are some alternatives, however, and in this post I will discuss the application of evaporative light scattering detection (ELSD) to flash purification.

Continue reading Detecting the undetectable in flash column chromatography, part 2

How do I purify my high boiling solvent reaction mixture using silica flash chromatography?

Many chemists today find they need to synthesize molecules at higher temperatures in order to force difficult reactions to proceed. Solvents such as DMF, DMSO, and NMP are commonly used in these reactions as they facilitate the use of the high reaction temperatures.  However, the same attributes that make these chemicals attractive as reaction solvents make compound recovery from them very difficult including flash column chromatography.  These high boiling solvents are typically polar and pose a challenge if purification is to be accomplished with normal-phase silica.

In this post I explore some options that enable purification of high boiling point solvated reaction mixtures through simple sample work-up.

Continue reading How do I purify my high boiling solvent reaction mixture using silica flash chromatography?

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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Does particle shape and/or particle size impact flash column chromatography load limits?

There are many factors which influence successful flash column chromatography. One of those factors is sample load which itself is influenced by things like selectivity, efficiency, dissolution solvent, and load technique. Several of these factors I have addressed in previous posts. Of these, selectivity and efficiency are specific to a media’s physical and chemical characteristics.

In this post I will show if particle size and/or particle shape can influence loading capacity.  Additionally, I will show the positive impact that surface area has on flash column chromatography purification.

Continue reading Does particle shape and/or particle size impact flash column chromatography load limits?

Non-aqueous (or nearly so) reversed-phase flash column chromatography – a nice alternative for purifying lipophilic compounds

For most organic and medicinal chemists, normal-phase flash chromatography is used to purify and isolate many types of organic compounds, most with some polar functional groups which help them retain on silica. However, some compound mixtures are water insoluble such as lipids, carotenoids, terpenes, tocopherols, polyaromatic and other hydrocarbons with minimal polar functionality.   These lipophilic compounds do not retain well on silica and do not dissolve readily in water making them really difficult to separate.

In this post I will talk about a technique called non-aqueous reversed-phase chromatography that can be very effective at separating and purifying very lipophilic compounds. Continue reading Non-aqueous (or nearly so) reversed-phase flash column chromatography – a nice alternative for purifying lipophilic compounds