Reversed phase flash chromatography is increasingly being utilized by peptide chemists to decrease purification time and efforts. The larger particles used in flash columns enable large crude sample loads and can lead to highly pure peptide samples despite lower resolution when compared to traditional HPLC methods. However, there are some situations where the purity achieved isn't sufficient.

Then what can you do?In today's post, I'll describe using a focused gradient to achieve higher purity peptides than is possible with a more traditional linear gradient.If there's anything constant about peptides, it's their unpredictability. Whether it's a synthesis that has gone totally sideways, or that one sequence that you just can't dissolve once it's cleaved from the resin, or worse yet that one peptide you can't quite get pure using a standard gradient.

In my experience, most groups start peptide purification with a standard gradient - whether that be a 2% per minute change in acetonitrile or a 1% per minute change in acetonitrile or something else, there has to be a starting point. Once this baseline is established, the gradient can be modified to enable purification of the peptide of interest.

The same general protocol is also employed when using reversed-phase flash chromatography for peptide purification. So how do you change the gradient to increase the peptide's purity?One thing is for sure, decreasing the slope of the gradient and extending the purification time doesn't work well in reversed-phase flash chromatography. Rather than increase resolution between peaks, as is often observed with HPLC, this strategy causes severe peak broadening and loss of peak differentiation and detectability.

So if extending the gradient doesn't work well, what other strategies can be used?I have written some about using a step gradient as a method to overcome the peak broadening issue mentioned above while enabling separation of single amino acid deletion sequences. However, this method can be difficult without some additional software help. As an alternative to a step gradient, a focused gradient could be used.

Reference

https://selekt.biotage.com/peptideblogs/how-to-use-a-focused-gradient-for-higher-purity-peptides-with-flash-chromatography