How to use a scouting column for your peptide purification

Whenever I synthesized a new peptide, I always ran a “scout run” – a small scale injection, usually with an analytical HPLC column – to both get an idea of the crude purity and also to identify a shorter, more optimal gradient for the actual purification.  This strategy is still probably fine when you want to use reversed phase flash chromatography for your purification strategy, but is there a better way?

In today’s post, I’ll discuss using a scouting column to screen gradient conditions prior to peptide purification with reversed phase flash chromatography.

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How to choose an ion pairing agent to improve your peptide purification

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?

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How does methanol as a mobile phase solvent impact peptide purification by reversed-phase flash chromatography?

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.

One alternative that is growing in popularity is the use of methanol in place of acetonitrile for reversed phase purification of small molecules.  Methanol is certainly less expensive, but is also a more environmentally-friendly solvent for use in purification applications.  But it’s use for peptide purification has not been widely adopted to date.  In today’s post, I’ll compare the purification efficiency of methanol when compared to acetonitrile for peptide purification by reversed phase flash chromatography.

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How to purify peptides using a step gradient in flash column chromatography

Flash chromatography can be a challenging technique for peptide purification due to the lower resolution achieved with large particles.  While some may see this as a disadvantage, the significantly greater loading capacity gives me reason to make this work. So how can I achieve the high purity levels often accessed using traditional reversed-phase HPLC methods?

In this post, I’ll discuss using a step gradient for peptide purification.  Step gradients are commonly used in normal-phase small molecule purification and typically improve the purification efficiency while reducing the overall purification time. Continue reading How to purify peptides using a step gradient in flash column chromatography

How does flow rate affect my peptide purification efficiency when using a small pore stationary phase?

In a previous post, I evaluated how flow rate can impact my purification efficiency using flash chromatography.  I noticed though, at high flow rates a significantly later elution time for my peptide.  I hypothesized that the increased pressure was driving the compound further into the pores, increasing the overall interaction with the stationary phase and causing the increased retention.  We know that the particle size and particle pore size impact resolution and purification efficiency, so how does flow rate play a role with a different stationary phase?

In today’s post I’ll evaluate several flow rates using a reversed phase stationary phase material with slightly larger diameter particles that possess significantly smaller pores.  The smaller pores should limit the access of the peptides to the stationary phase and negatively impact the purification.

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