Mass-directed purification, whether with a preparative HPLC or a bench-top flash system, is quickly gaining interest in the peptide purification space. The simple fact is that using a specific mass, rather that UV absorbance, to trigger fraction collection allows for greater confidence in the identity of the collected fraction. Importantly though, this technique can also reduce your time required for purification, by significantly reducing or even eliminating the need for secondary mass analysis of each collected fraction.
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?
In peptide purification, sample loading onto the column is rarely considered. Most, if not all, HPLC instruments come equipped with a sample injection loop which demands a liquid injection of the sample for purification. If you decide to use flash chromatography to purify your peptides though, liquid injection is no longer the exclusive method for sample introduction to the column. Alternatively, dry loading crude material is a strategy often used in small molecule purification, particularly when sample solubility concerns arise.
Chemical synthesis of peptides, and even proteins, offers the possibility to expand the functionality and stability imbued by nature. However, chemical synthesis of very long peptides and small proteins remains today an exceedingly difficult task. Several ligation strategies have been developed that help to alleviate this challenge. These strategies though, require a purified, yet fully protected peptide fragment.
"How can I take a two hour purification using old-school chromatography, and shorten it?" was the question Dr. Aaron Muth at St. John's University in New York asked himself. "By using the Biotage Isolera, I could take it down to 10 or 15 minutes."