Promix™ Columns - Two Dimensional Separation of Proteins & Peptides

Significance of the topic

Advances in proteomics have driven the need for highly selective and high-capacity chromatographic separations of proteins and peptides. Complexity and structural similarity among biomolecules often limit traditional ion-exchange or reverse-phase methods. Two-dimensional HPLC approaches that combine orthogonal interactions address these challenges by enhancing resolution, enabling effective purification for analytical and preparative workflows.

Objectives and overview of the study

This application note evaluates Promix™ columns featuring integrated ion-exchange and reverse-phase functionalities. Key aims include:

• Demonstrating separation of insulin analogs differing by a single amino acid.
• Resolving complex peptide mixtures spanning small oligopeptides to larger proteins.
• Assessing the impact of buffer type and ionic strength on selectivity.

Methodology and instrumentation

Promix MP, SP and AP columns of various dimensions (50×3.0 mm to 250×4.6 mm) were used on standard HPLC systems. Mobile phases comprised acetonitrile gradients combined with volatile acids (formic, phosphoric, perchloric, sulfuric) or ammonium salts (formate, acetate) at pH 2.0–4.0. Flow rates were maintained at 1.0 mL/min; detection was performed at 210 or 270 nm. Injection volumes ranged from 5 to 100 µL depending on sample concentration.

Main results and discussion

• Insulin analogs separating normal human insulin, Humalog, and Lantus achieved baseline resolution (Rs up to 1.7) by tuning ionic (pI differences) and organic gradients.
• Complex mixtures of five peptides (0.6–29 kDa) were fractionated sequentially: small peptides eluted early on Promix MP and then re-analyzed on SP for deeper resolution.
• Buffers with differing ionic strengths and counterions significantly altered elution profiles, demonstrating customizable selectivity via independent adjustment of organic and salt gradients.
• Secretin and β-secretin separation illustrated the method’s sensitivity to subtle sequence variants, with adequate resolution even under mixed gradient conditions.

Benefits and practical applications

The Promix two-dimensional approach offers:

• Enhanced selectivity for homologous peptides and proteins by exploiting combined ionic and hydrophobic interactions.
• High peak capacity that surpasses single-mode columns.
• Scalability from capillary to preparative formats, facilitating both analytical profiling and bulk purification.
• Compatibility with LC-MS workflows through use of volatile buffers and acids.

Future trends and opportunities

Emerging demands in proteome profiling, biopharmaceutical characterization, and quality control will benefit from further integration of multidimensional separations. Future developments may include automation of fraction collection and on-line MS coupling for real-time identification. Customizable stationary phases tailored to post-translational modifications and intact protein analysis represent additional growth areas.

Conclusion

Promix columns deliver robust two-dimensional separations by seamlessly combining ion-exchange and reverse-phase interactions. They achieve high resolution for challenging protein and peptide mixtures, offering flexibility for research and industrial applications, and compatibility with MS detection.

Instrumental setup

• Columns: Promix MP, SP, AP (50×3.0 mm to 250×4.6 mm)
• Mobile phases: Acetonitrile gradients (0–70%) with TFA, formic, phosphoric, perchloric, sulfuric acids or ammonium formate/acetate (5–30 mM) at pH 2.0–4.0
• Flow rate: 1.0 mL/min
• Detection: UV at 210 or 270 nm
• Injection volume: 5–100 µL