Separation of Proteins and Peptides on Discovery BIO Wide Pore (300Å) C18, C8 and C5 Phases

By: Hillel Brandes and Tracy Ascah, Reporter US Volume 27.3

Hillel Brandes and Tracy Ascah


When performing protein and peptide separations, scientists prefer HPLC bonded phases to be stable and reproducible while exhibiting high resolution. Discovery® BIO Wide Pore C18, C8 and C5 were specifi cally developed for those needs. The C18 and C8 phases demonstrate high resolution peptide mapping and purifi cation. The C5 phase exhibits enhanced stability and is excellent for protein separation. All of the phases show high lot-to-lot reproducibility.

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Peptide Mapping and Purification with Discovery BIO Wide Pore C18 and C8 phases

Peptide and protein chemists use C18 or C8 phases for peptide mapping and purifi cation. However, problems of peak tailing or low resolution may be encountered due to secondary chromatographic effects. Some unnecessary active sites, such as active silanol groups and metal impurities, may still remain on the surface. This is especially critical for peptide mapping because it often requires resolving more than a hundred peaks in one hour. Figure 1 presents a comparison of a peptide map with well resolved peaks on Discovery BIO Wide Pore C18 phase vs. the same application on a prominent competitor’s column.

Figure 1. Efficiency of Discovery BIO Wide Pore C18 Gives Higher Resolution Tryptic Maps than Competitive C18 Phase


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Protein Analysis and Purification with C5 Phase

In protein analysis and purification, possible sample denaturation is a consideration. For this reason, we would choose to use a short alkyl chain phase, such as C4, for the application. This may reduce the length of time the sample is retained on the column and lessen the amount of the stronger organic solvent needed. However, C4 is very susceptible to acid hydrolysis and may not remain stable over time. This problem is overcome on the Discovery BIO Wide Pore C5. It demonstrates almost identical selectivity to C4, but is substantially more stable, translating into longer column life. Figure 2 compares the stabilities between Discovery BIO Wide Pore C5 and a conventional C4 phase.

Figure 2. Stability Comparison of Discovery BIO Wide Pore C5 and a Conventional C4 HPLC Column


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Reproducibility of the Phase

Reproducibility is another key factor to consider when selecting a column, and is generally discussed in terms of run-to-run, column-to-column, and lot-to-lot (or batch-tobatch). Variation in run-to-run reproducibility is generally very low, provided the HPLC instrumentation is well maintained and the bonded phase is stable. Differences in column-to-column reproducibility within the same lot are also negligible with today’s standardized column packing and manufacturing processes. Rather, most reproducibility problems arise from lot-to-lot variances. Factors influencing lot-to-lot reproducibility include differences in silica lots, reagent lots, other processing materials, and operators. However, when strictly controlled, those effects can be greatly minimized. Figure 3 presents the results of a peptide test mix performed on three lots of Discovery BIO Wide Pore C5. The RSD of the retention time of last peak is less than 2%.

Figure 3. Results of a Peptide Mix on Three Different Lots of Discovery BIO Wide Pore C5

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To separate proteins and peptides by reversed phase chromatography, consideration should be given to column resolution, stability and reproducibility. Discovery BIO Wide Pore C18 and C8 have been developed specifi cally to address these issues. In protein separations, C5 is generally a better choice than C4 because it maintains the advantages and selectivity of a short alkyl phase while exhibiting superior stability.

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Discovery BIO Wide Pore Characteristics

Phases: C5, C8 and C18
Particles: 3, 5 and 10 μm spherical,
high-purity silica
Pore Diameter: 300 Å
Columns: capillary to preparative dimensions

The most popular dimensions of Discovery BIO Wide Pore are listed below. For the complete listing, including capillaries and guard columns, please call our Technical Services or visit

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