Promix - Enter a New Era in Biomolecule Analysis with Promix Columns

Importance of the Topic

The analysis of peptides and proteins requires high selectivity and peak capacity to resolve closely related biomolecules and improve sensitivity in LC-MS detection. Mixed-mode chromatography, which combines ionic and reversed-phase interactions in a single column, addresses limitations of traditional methods by offering orthogonal retention controls and scalable formats.

Objectives and Study Overview

This work introduces Promix™ mixed-mode columns as a versatile solution for biomolecule analysis. It demonstrates their performance across applications—synthetic peptide purification, insulin analog separation, complex mixture profiling, protein digests, and both small and large peptide separations—and provides guidance for column selection and method optimization.

Instrumentation Used

• Promix MP, SP, AP and LP mixed-mode columns (various pore sizes and formats).
• HPLC system with UV detection at 210–270 nm.
• Mobile phases: acetonitrile (MeCN), trifluoroacetic acid (TFA), formic acid, ammonium acetate or formate buffers (pH 2–4).

Methodology and Instrumentation

Promix columns feature an ionic functional group for pI-dependent interactions and a hydrophobic chain for organic modifier–controlled retention. Independent gradients of buffer strength and organic content enable isocratic or gradient separations. Columns range from capillary to preparative scale and support multiple separation modes: MeCN gradient with fixed buffer, buffer gradient with fixed MeCN, or dual gradients.

Key Results and Discussion

• Isocratic separation of a synthetic peptide and its impurity demonstrated clear resolution under mixed-mode conditions superior to reversed-phase alone.
• Insulin analogs differing by single amino acid pairs were resolved based on slight pI differences; resolution improved with higher buffer pH.
• A two-step protocol using Promix MP then SP columns achieved comprehensive profiling of complex mixtures spanning very small to large peptides.
• Mixtures of Humalog and Lantus insulins exhibited tunable selectivity through systematic adjustments of MeCN and TFA gradients.
• Reinjection of digest fractions from a reversed-phase column onto Promix MP extended elution windows and enhanced peak capacity.
• Small and large peptide test mixtures yielded distinct separation patterns, validating column versatility across size ranges.

Benefits and Practical Applications

• Enhanced selectivity and peak capacity enable discrimination of biomolecules differing by minimal structural changes.
• One-column 2-D HPLC simplifies workflows and reduces instrument complexity.
• Scalable formats support applications from nano-LC proteomics to preparative peptide purification.
• Compatibility with MS-friendly buffers improves sensitivity and throughput in biomolecule characterization.

Future Trends and Opportunities

Mixed-mode chromatography is poised to integrate with advanced mass spectrometry platforms and automated multidimensional workflows. Continued development of novel stationary phases and online gradient strategies will expand separations for post-translationally modified proteins and other challenging biomolecules.

Conclusion

Promix mixed-mode columns deliver flexible, high-resolution separations by decoupling ionic and hydrophobic retention mechanisms. Their scalability and orthogonal selectivity make them a powerful tool for analytical and preparative workflows in proteomics, pharmaceutical development, and quality control.