A Comparison of Superficially Porous Particle-based Column Chemistries for Peptide Mapping

by | Jan 9, 2024

Explore in-depth insights into the performance of the C18 and alternative selectivities in peptide separation.

This article from issue 15 of the Analytix Reporter investigates the performance of three superficially porous particle chemistries—cyano, phenyl-hexyl, and C18 from the BIOshell™ line of U/HPLC columns—in the separation of peptides and peptide mapping. The study evaluates key parameters such as peak width, peak capacity, resolution, and theoretical plates, using a system suitability test mix (MSRT1) and a tryptic digest of the monoclonal antibody reference material NISTmAb.

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Superficially porous particles (SPP) have proven themselves as an efficient alternative to fully porous particles in HPLC separations. Higher efficiency per backpressure unit is achieved with these particles, in comparison with fully porous particles, and the advantages of this particle technology have been reported in the literature. These higher efficiencies are due to shorter diffusion paths within, and narrower particle size distributions of the SPP.

In this application, we compare three different superficially porous particle chemistries, from the BIOshell™ line of U/HPLC columns, in terms of their performance in the separation of peptides and peptide mapping. The quality parameters evaluated include peak width at half maximum (FWHM, full width half maximum), peak capacity, resolution between selected peak pairs, and theoretical plates (N).

For system suitability testing, a mixture of synthetic peptides in the MSRT Calibration Mix (MSRT1)was first used to compare the cyano, phenyl-hexyl, and C18 bonded phases, prior to performing the same comparisons with a tryptic digest of the monoclonal antibody reference material NISTmAb, a humanized IgG1k monoclonal antibody.


The system suitability mix, MSRT1, was prepared according to the instructions on the data sheet but with a final acetonitrile concentration of 1.6%. The injection volume was 10 µL.

Digestion of NISTmAb reference material 8671 was performed with a low artifact digestion buffer (EMS0011) using instructions provided in the product information sheet. To look for oxidized and deamidated forms of peptides, a digestion was also performed using overnight digestion in ammonium carbonate buffer. In this way, higher amounts of oxidized methionine and deamidated asparagine were generated on some peptides to evaluate chromatographic separations.

Instrument and gradient conditions are found in Tables 2-4 in the full article.


A system suitability test mix is a recommended way to monitor the performance of a chromatographic system prior to submitting valuable samples for analysis. MSRT1 is a mix of 14 isotopically labeled peptides whose sequences are shown in Table 5 in the article. Each peptide is labeled with an isotopically labeled form of either [13C6, 15N1] leucine, [13C6, 15N2] lysine, or [13C6, 15N4] arginine (shown in brackets).

Injection of this mix on each of the three columns (Figure 1 in the article) indicates increasing retention of peptides in the order of cyano, phenyl-hexyl, and C18 under identical chromatographic conditions.

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As expected, the C18 chemistry provided the best retention of peptides, with the phenyl-hexyl phase next, followed by cyano. Peak widths generally followed the same sequence with C18 again performing the best and, therefore, providing the highest peak capacity, and plates. Regarding the resolution of three selected peak pairs, it is apparent that the different phases do offer slight differences in selectivity so that, in some cases, a cyano or phenyl-hexyl chemistry may outperform a C18 phase. 

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*The life science business of Merck operates as MilliporeSigma in the U.S. and Canada.

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