How does media pore size impact peptide resolving power?

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.

The influence of pore size is well know in the HPLC column community, as demonstrated by the vast array of columns available that vary in both particle size and pore size, but is not commonly discussed in the flash column chromatography community.  Recently, I was asked to evaluate some new C18-functionalized media that has a pore size of approximately 300Å and I jumped at the opportunity to identify the differences.

For this evaluation, I compared the resolving power of the Biotage® SNAP Ultra C18 (90Å pore) to that of the new Biotage® SNAP Bio C18 (300Å pore).  For this comparison, I felt it critical to use crude peptide from a single synthesis and fortunately I had recently completed a large scale synthesis of ACP(65-74).  After conducting analytical RP-HPLC-MS, I calculated the crude sample to be about 36% pure; this sample contains a high concentration of residual protecting groups, Figure 1.

Figure 1:  Crude analytical HPLC chromatogram for peptides purified below.

I first loaded about 50 mg of my peptide dissolved in DMSO onto the Biotage® SNAP Ultra C18 12 gram cartridge for purification using a somewhat optimized gradient, Figure 2.

Figure 2:  Chromatogram resulting from 50 mg ACP in 300 uL DMSO loaded onto a 12 gram Biotage® SNAP Ultra C18 cartridge.  The peptide is purified using a gradient that runs from 10% to 40% acetonitrile in water, both modified with TFA.

The peak shape is certainly not gaussian, suggesting the presence of co-eluting molecules.  I monitored the elution by MS, but collected the peaks based on UV absorbance.  With these strategies as a guide, I combined fractions 4-6 (center yellow peak), concentrated them with the Biotage® V-10 Evaporation system and re-injected the peptide on my analytical HPLC-MS where I found the sample to be about 77% pure, Figure 3.  Not the best results…

Figure 3: Analytical HPLC of ACP peptide purified using the Biotage® SNAP Ultra C18 cartridge.

For comparison sake, I injected approximately 50 mg of crude ACP dissolved in DMSO onto a Biotage® SNAP Bio C18 cartridge using the exact same gradient as the previous injection, Figure 4.

Figure 4: Chromatography of 50 mg crude ACP loaded on a Biotage® SNAP Bio C18 cartridge.

Results from the larger pore media show a distinct leading shoulder which was sufficiently resolved to trigger a fraction change.  Using the MS and UV traces as a guide,  I combined fractions 4 and 5 (middle yellow peak), quickly concentrated them with the Biotage V-10 evaporation system and reinjected onto the analytical HPLC-MS as before. Using the analytical HPLC-MS, I calculated the sample to be approximately 92% pure, Figure 5.  A significant improvement over the previous sample!

Figure 5: Analytical HPLC chromatogram for ACP purified using the Biotage® SNAP Bio C18 cartridge.

As a follow up experiment, I decided to reinject the peptide originally purified using the Biotage® SNAP Ultra C18 onto the new Biotage® SNAP Bio C18.  The second chromatogram clearly shows the expected leading shoulder, which is still resolved even at significantly lower loading levels, Figure 6.

Figure 6: Chromatogram for second purification of ACP using the Biotage SNAP Bio C18 cartridge.

Analysis of the resulting analytical HPLC  clearly indicate that the second purification effort did in fact improve the overall purity (up to 93% from the original 77% purity), Figure 7,

Figure 7: Analytical HPLC chromatogram resulting from a second purification using the Biotage® SNAP Bio C18 of ACP originally purified using the Biotage® SNAP Ultra C18 cartridge.

The dramatic increase in purity resulting from the purification attempt suggests that the improvement in purification is due to the increased particle pore size, rather than an artifact result from amount of material loaded on the column.  The larger pore size enables greater molecular accessibility to the stationary phase alkyl chains inside the pores, thereby increasing the probability that the molecule of interest will have maximal interaction with the stationary phase.  For flash chromatography, it is clear that the larger pore size yields a higher purity peptide product in a single injection.  I expect that this will also follow for larger peptides as well, but that’s for a later discussion.

What are your experiences in peptide purification using stationary phases with a variety of pore sizes?


Published by

Elizabeth Denton

Elizabeth Denton is the Peptide Application specialist for Biotage and handles all things peptide synthesis and workflow related, in addition to troubleshooting problematic syntheses. Elizabeth recently began her career at Biotage after working as a peptide scientist for a small biotech company. Elizabeth earned a bachelors degree in Chemistry from Arizona State University and a Ph. D. in Chemistry from Yale University with a focus in Chemical Biology. Throughout her thesis work, Elizabeth focused on projects in which peptides, containing both natural and unnatural amino acids, were designed to target and activate natural proteins.

4 thoughts on “How does media pore size impact peptide resolving power?”

  1. Hi Elizabeth,

    Thanks for the post. I have a few questions if you don’t mind.

    I’m curious, in this case you had a 10 amino acid peptide with a mass (I am assuming) around 1500 Da which doesn’t seem especially large. I was under the impression that pore size of 100angstroms is appropriate for molecules up to 2000 Da or so, when dealing with HPLC. At what molecular weight do you expect you will start to see a significant improvement in separation moving to a larger pore size? Would you expect to see a much greater effect with a larger molecule with MW of 5000 or 10000 Da?

    Another question, would you expect the separation to be influenced strongly by the properties of the molecule, ie. hydrophilicity/phobicity, compact or diffuse structure, pegylated compounds, etc., and how so in general?


    1. Hello Graham,
      Thanks for your questions! You are correct that for typical HPLC purifications a pore size of 100 angstroms is often used for peptides up to about 3000 Da. Typically in HPLC purifications, a larger pore sized media is recommended for peptides or proteins greater than about 4000 Da. I would expect similar improvements for large peptide samples as well. Bear in mind that larger synthetic peptides often come with more closely eluting contaminants which will likely not be resolved with flash chromatography. I saw a significant improvement in the quality of purification when I moved to the larger pore media for the 10 amino acid peptide which was somewhat surprising. Remember, flash chromatography is a lower resolution technique due to the larger particle size and I suspect that the greater resolution observed for such a small peptide is due to greater interaction with the C18 alkyl chains within the larger pores.

      I absolutely expect any chromatography to be influenced by the properties of the molecule. For reversed phase chromatography, the molecules are separated via a partitioning mechanism between the stationary phase and the mobile phase. Typically, a hydrophobic molecule would be highly retained and difficult to elute from the stationary phase. However, if that molecule was folded in a manner that shields a substantial proportion of the hydrophobic surface, the molecule would be significantly less retained by the stationary phase. While this is a somewhat simple example, all of the characteristics you described above will absolutely impact chromatography to varying degrees.

      To answer your second question, I absolutely expect chromatography to be influenced by the properties of the molecule.

  2. Great post. I’ve always wondered how pore size affects flash chromatography. I do small molecules so I presume in this case it doesn’t matter that much. BTW. What was your product recovery after these injections? Ohh and Fig 7 is mislabelled as Fig 6.

    1. Hello Tomasz,
      Thank you for your interest (and for catching my error!). Stay tuned for a future post studying the effects of pore size for small molecule purification. As to your recovery question, I recovered about 75% of what I expected from the SNAP Ultra C18 cartridge (calculated based on recovered product purity) and about 85% of what I expected from the SNAP Bio C18 cartridge.

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