In a previous post, I evaluated how flow rate can impact my purification efficiency using flash chromatography. I noticed though, that my peptide eluted significantly later with high mobile phase flow rates. I hypothesized that the increased pressure (caused by higher flow rates) 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?
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.
We've all used mass spectrometry to characterize our synthetic peptides. It's often used to confirm that the peptide was in fact synthesized, then again as part of the purification process to make sure that we're collecting the correct peak. But how many of you had the opportunity to use in-line mass spectrometry as an integral component during the purification itself?
In today's post, I'll highlight some of the advantages to using in-line mass mass spectrometry for purification of peptides using reversed phase flash chromatography.
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?