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.
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.
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.
In the past, when I have 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 recommended 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.
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.
Whether it’s the bonded stationary phase, particle size, or even particle pore size, scientists today are offered a plethora of choices when it comes to reversed phase HPLC columns. An often acknowledged concern in the peptide community though is peptide recovery from reversed phase purification efforts, particularly for precious peptide mixtures. But how is peptide recovery impacted when you use reversed phase flash chromatography for purification?
In today’s post, I’ll compare recovery levels for two peptides that differ in length as well as crude purity using reversed phase flash chromatography. In addition to comparing two peptides, I’ll also evaluate how recovery is impacted by altering the mobile phase pH.
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?
As a peptide chemist, I was trained to purify my peptides with reversed-phase HPLC, just as many a peptide chemist before me. Despite the hundreds of hours I’ve logged in front of an HPLC, injecting samples and collecting peak fractions, I can’t imagine using any other method to purify my freshly synthesized and cleaved peptides. In fact, you’d be hard pressed to convince me to try something else. But here I am, trying something new. Wish me luck!
In this post, I’ll describe my experiences using flash chromatography to purify a new peptide sample.
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.
There are several strategies often employed to improve peptide purity achieved using reversed phase HPLC. These strategies can include, changing column length, particle size, particle functionality (C4 vs C18). I have experimented a bit with some of these criteria while purifying peptides using reversed phase flash chromatography but one obvious change that I have not yet explored is the length of column.
In today's post, I'll explore how the length of the cartridge affects the overall resolution and purification efficiency using reversed phase flash column chromatography.