Are reversed-phase flash chromatography columns designed for aqueous solvents necessary?

When it comes to the purification of polar, water-soluble compounds reversed-phase chromatography is the most commonly used approach. However, because of strong stationary phase – mobile phase repulsion forces, the use of highly aqueous (90-100% water) solvent systems has been shown to provide less retention than needed.  This issue has led to the development of “aqueous compatible” reversed-phase media.

In this post I explore if these types of phases are actually needed by looking at the separation of some very polar and low log P compounds using a “traditional” C18-bonded silica.

Continue reading Are reversed-phase flash chromatography columns designed for aqueous solvents necessary?

How does solvent choice impact flash column chromatography performance?

Selectivity and solvent strength are the most important factors that determine success or failure of a chromatographic separation. These two independent dynamics apply to both normal- and reversed-phase chromatography and should be optimized, especially when high fraction purity is needed.

In this post I will discuss the impact that elution solvent choice has on both normal- and reversed-phase purification. Continue reading How does solvent choice impact flash column chromatography performance?

Are there advantages to stacking flash chromatography columns?

When it comes to isolating a compound from a mixture at maximum purity there are many options available through flash column chromatography. In previous posts I have addressed using smaller particle media, higher surface area media, and step gradients to achieve this goal.

In this post I will discuss how stacking columns in series may help improve separation quality. Continue reading Are there advantages to stacking flash chromatography columns?

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.

Continue reading How does media pore size impact peptide resolving power?

In-line mass detection to find the undetectable in flash column chromatography

In previous posts I have discussed some options and techniques to improve detection of poorly absorbing or UV-transparent compounds – by wavelength focusing and by evaporative light-scattering (ELS).

In this post I will talk about a third alternative technique – using an in-line mass detector. Continue reading In-line mass detection to find the undetectable in flash column chromatography

Benefits of acetonitrile over methanol in normal-phase flash column chromatography

When it comes to the purification of polar organic compounds many chemists turn to normal-phase flash chromatography with dichloromethane and methanol as the mobile phase. This solvent system often can be challenging to optimize due to methanol’s high polarity and protic chemistry.

I have found that acetonitrile can often replace methanol as the polar modifier in DCM-based solvent systems.  In this post I will show an example where this is true. Continue reading Benefits of acetonitrile over methanol in normal-phase flash column chromatography

Pushing flash column chromatography loading limits

A question I hear a lot from chemists is “how much can I load”. The answer is always “it depends on your separation quality”.  At that point I begin asking about the TLC data and purification goals. Purification goal setting should be your first step and the question to answer is – what do I need this purification to achieve? Is the goal high purity, high yield, or some combination.  Remember, you will typically sacrifice purity for high yield and yield for high purity so optimization is an important consideration.

In this post I will discuss the results of a study I conducted where I continually increased the crude sample load until my target compound purity fell below specific targeted levels to understand the real loading limitation. Continue reading Pushing flash column chromatography loading limits

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.

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

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