LC columns and accessories for biomolecules

Importance of the topic

Effective separation of proteins, peptides, oligonucleotides and complex carbohydrates is fundamental for research, quality control and biopharmaceutical development. Variability in molecular size, charge and hydrophobicity demands tailored stationary phases and modes of chromatography to achieve accurate identification, quantitation and characterization of biomolecules.

Objectives and overview of the article

The document provides a comprehensive guide to liquid chromatography columns and accessory chemistries designed specifically for biomolecular analysis. It aims to help analysts select appropriate reversed-phase, ion-exchange, size-exclusion, hydrophobic interaction, affinity and HILIC phases based on sample type and application, from intact protein separations to oligonucleotide purification.

Used methodology and instrumentation

The guide covers multiple chromatographic modes implemented on HPLC, UHPLC and nano-LC platforms, with detectors such as UV, fluorescence and mass spectrometry. Major column families include:

• Silica-based fully porous and solid-core reversed-phase and ion-exchange columns (Acclaim, BioBasic, Accucore)
• Wide-pore and polymer-coated SEC columns (BioBasic SEC, MAbPac SEC-1)
• Monolithic polymer columns for high-flow, high-resolution separations (ProSwift RP, IEX, affinity)
• Strong and weak ion-exchange phases for cationic and anionic biomolecules (ProPac, MAbPac, DNAPac)
• HIC and affinity media for variant and aggregate analysis (ProPac HIC, MAbPac HIC, Protein A, IMAC, ConA)
• HILIC and mixed-mode phases for glycan and polar compound analysis (Accucore Amide-HILIC, GlycanPac AXH-1)

Main results and discussion

Key performance highlights include:

• Reversed-phase polymeric wide-pore columns (MAbPac RP) tolerate extreme pH (1–14) and high temperature (up to 110 °C), enabling robust denaturing protein separations.
• Dense bonding chemistries on BioBasic and Acclaim columns deliver reproducible C18, C8 and C4 separations with sub-2-µm particles for high throughput and MS compatibility.
• Pellicular and monolithic ion-exchange columns (ProPac, MAbPac, ProSwift) achieve single-residue resolution of protein charge variants with fast mass transfer and moderate capacity.
• Size-exclusion phases spanning 60 Å to 1000 Å pore sizes support accurate molecular weight determination, aggregate profiling and sample cleanup.
• Hydrophobic interaction columns (ProPac HIC, MAbPac HIC) offer high resolution of mAb variants and fast method development for analytical and preparative applications.
• Affinity materials (Protein A, IMAC, ConA) enable selective enrichment, titer determination and glycan profiling in complex biological matrices.
• Strong anion-exchange and reversed-phase DNAPac and DNASwift columns resolve full-length oligonucleotides, failure sequences and large dsDNA fragments up to 10 000 bp.
• HILIC and mixed-mode glycan columns (Accucore Amide-HILIC, GlycanPac AXH-1) deliver high peak capacity and reproducible separations of fluorescently labeled or native glycans.
• Nano and capillary PepMap and EASY-Spray columns support high sensitivity LC-MS/MS workflows for proteomics and biomarker discovery.

Benefits and practical applications

These column chemistries provide:

• High resolution and peak capacity across diverse biomolecules
• Robust operation under extreme pH and temperature for denaturing and native analysis
• Fast method development and high throughput via monolithic media and sub-2-µm particles
• Compatibility with LC-UV, fluorescence and high-resolution mass spectrometry
• Scalable workflows from analytical to preparative and semipreparative scales

Future trends and possibilities for use

Emerging directions include integration of two-dimensional separations, further miniaturization for single-cell analysis, tailored monolith and solid-core designs for ultra-fast gradients, and AI-driven method optimization. Continued expansion of biocompatible stationary phases will support next-generation biologics characterization and personalized medicine applications.

Conclusion

Thermo Scientific’s comprehensive portfolio of silica and polymeric columns addresses the unique challenges of biomolecular separations. By matching stationary phase chemistry and configuration to target analytes and detection methods, analysts can achieve reliable, high-throughput workflows for protein, peptide, oligonucleotide and glycan analysis across research and industrial laboratories.