Synthesizing hydrophilic peptides can be relatively straightforward, especially when compared to the trials and tribulations encountered when synthesizing hydrophobic peptides. However, purifying hydrophilic peptides using standard reversed-phase chromatography can be a challenge. In a previous post, I encountered this problem during my analytical HPLC analysis of crude peptide mixtures.
In this post, I will discuss the use of ion pairing agents to increase the perceived hydrophobicity of your peptide, increasing the column retention and enabling a smooth purification of your hydrophilic peptide.
A few weeks ago I synthesized Arg-8 using a variety of different coupling strategies. However when it came time to analyze the crude peptide samples, the peptide always eluted with the injection front. So when it came time to purify the samples, I had to find a different strategy to ensure the peptide would be retained by the column and successfully purified. I had a professor that once said, “an hour in the library will save you days at the bench” and with that in mind I turned to my favorite peptide HPLC book edited by Hodges and Mant.
In this book there are several chapters discussing the use of ion pairing agents in peptide purification. I decided to put this strategy to practice in my efforts to use flash column chromatography to purify my Arg-8 peptides. Of the variety of choices commonly used, I chose to compare the efficacy of TFA, heptafluorbutyric acid (HFBA), and perfluorpentanoic acid (PFPA). The most significant advantage of these additives over others is that they are all still somewhat volatile, enabling the use of mass detection without concerns for damaging the instrumentation.
I first injected Arg-8, dissolved in acidified water, onto a Biotage® SNAP Bio C18 cartridge using unmodified water and acetonitrile for my mobile phase, Figure 1. While in practice this is not something I would ever recommend, for the purposes of this investigation I wanted a baseline measurement of retention.
And as expected, based on the analytical challenges, the peptide is barely retained by the cartridge (blue peak). While there is some degree of purification occurring here, it is clear that improvements can still be made.
For the second purification, I injected crude Arg-8 onto the same Biotage SNAP Bio C18 cartridge, after equilibration with mobile phase modified with 0.1% TFA, Figure 2; this is much more reminiscent of standard peptide purification strategies.
Using this strategy, the trifluoracetate anion pairs with the cationic sidechains, increasing the overall hydrophobicity and retention of the peptide on the C18 cartridge. In this case, it appears that the trifluoracetate ion pair will enable sufficient purification from any contaminants, but what are the effects of other ion pairing agents?
To further explore this question, I injected my crude Arg-8 on to the same Biotage SNAP Bio C18 cartridge, pre-equilibrated in mobile phase modified with heptafluorbutyric acid (HFBA), Figure 3.
The ion pairing strength of HFBA is similar to that of TFA, but the hydrophobicity of the anion is significantly greater, increasing the peptide retention to approximately 45% acetonitrile when compared to approximately 20% with solvents modified with TFA. You’ll likely notice that the solvent gradient has been altered to accommodate the increased cartridge retention of the peptide. Importantly though, several contaminants are also resolved and removed that were not separable with the TFA additive.
The increased peptide retention was even greater when attempting to purify Arg-8 using solvents modified with 0.1% perfluoropentanoic acid (PFPA), Figure 4. This should be expected, considering the pentanoic acid contributes more hydrophobic content when compared to the butyric acid and acetate additives.
In addition to increased peptide retention, the purification effort results in an Arg-8 sample with greater purity than the two previous purification attempts. All solvent gradients utilized in this exploration maintained the same linear gradient (50% change in 10 CV), highlighting the importance of changing mobile phase additives for peptide purifications.
While this purification effort utilized a peptide that contains many positively charged side chains, this strategy is particularly effective for crude peptide mixtures containing molecules that differ by a single cationic charge. The greater hydrophobic contribution of the PFPA can enable separation of similar peptides not resolvable with the more commonly used TFA additive.
Have you ever tried alternative ion pairing agents in your peptide purification efforts?