Protein Separation Technology UPLC and HPLC Columns

Waters is a world leader in the research, development, and commercialization of C₁₈, reversed-phase columns for many applications. However, the separation of large molecular weight biological compounds can be challenging and be significantly improved by changing the physical properties of the column packing material. For more than 15 years, chromatographers have benefited from performance characteristics that Waters reversed-phase columns have provided for protein separations. Now see how Waters Ethyl Bridged Hybrid (BEH) Particle Technology can help overcome performance limitations experienced using 100% silica-based materials for challenging protein separations.

• 300Å pores
• C₄ ligand
• Minimal undesirable secondary interactions
• Minimal detectable carryover at elevated temperature separations

300Å, C₄ Columns Developed for Protein Chromatography

The separation of a standard protein mixture shows that the BEH300 C₄ column can be used with proteins representing a wide range of properties. This sample includes proteins with high and low isoelectric points, molecular weights from 12-94 kDa, and that elute anywhere from 20-55% acetonitrile.

Waters invests significant research and development time to deliver solutions that help organizations address today's separation challenges. As a primary manufacturer of separations media, our ability to consistently deliver reliable, quality products, and support is second to none in the industry. Discover how our BEH300 C₄ columns supported by our world-class R&D, applications, and distribution organizations, help make the "Science of What's Possible" a reality.

• Well characterized, state-of-the-art bonding procedures
• Low pH and high temperature stability
• Quality-control tested with diverse protein mixture
• Consistent batch-to-batch reproducibility
• Available 1.7 μm particles for UPLC and nano UPLC applications

BEH300 C₄ vs. Competitive Column Stability Under Protein Separation Conditions

Overlay of 6 injections on XBridge^TM BEH300 C₄ vs. competitive 100% silica, 300Å C4 column. Column stored with 0.1% TFA at 90 °C between protein sample injections at times 0, 5, 10, 40 and 60 hours.

Rigorous Manufacturing and Testing Procedures Help Ensure Consistent Batch-to-Batch Performance

Outstanding Batch-to-Batch Performance Consistency when Tested with a Complex Protein Test Mixture.

In 2006, Waters introduced UPLC^® Technology that demonstrated outstanding performance enhancements in resolution and speed compared to use of traditional HPLC. Our new BEH300 C₄ material is available in two particle-sizes for easy transfer an HPLC protein separation method using 3.5 µm particles to the benefits of ACQUITY UPLC^® Technology using 1.7µm particles. In addition, BEH300 C₄ material can be effectively run with mass spectrometry compatible eluents for collection of the information rich data possible using this advanced detection technique.

• Easily scaled 3.5 μm HPLC to 1.7 μm UPLC for enhanced separations
• Effective with MS compatible eluents to address the need for advanced detection techniques

                    1.7 μm Particle Technology Benefits to Complex Protein Separations

Use of 1.7 μm particle technology yields improved resolution within mAb heavy chain fragment. Note that the full performance benefits of the ACQUITY^® BEH300 C₄, 1.7 μm column will only be realized with the low system volume and low detector dispersion of an optimized ACQUITY UPLC system.

Compared to many 100% silica-based reversed-phase columns, BEH300 C₄ offerings can be successfully used with MS friendly eluents.

Waters Protein-Pak^TM Hi Res IEX family of columns were developed to assist in the UPLC characterization of recombinant proteins and monoclonal antibodies destined to be used as therapeutics.  The non-porous, high-ligand density particles in these columns give higher resolution for high molecular weight proteins compared to traditional porous IEX particles. In addition, the particles in Protein-Pak^TM Hi Res Ion-Exchange columns are surface modified with a multi-layered network of ion-exchange groups (Sulfopropyl, Carboxymethyl or Quaternary ammonium), which assists in the effective binding of charged proteins. This allows for high sample loading capacities and component resolution while minimizing column fouling. 

• Innovative particle and ligand bonding processes yield protein binding capacities typically seen with porous IEX materials while delivering component resolution of large proteins usually observed with non-porous IEX columns.
• Quality-control tested with protein samples to help ensure consistent batch-to-batch performance.
• Columns containing hydrophillic, polymer-based IEX particles can be effectively cleaned with aggressive wash protocols.
• Column history and performance optimized when used on an ACQUITY^® UPLC System.

• Determines aggregation levels in therapeutic monoclonal antibodies up to 10 times faster than traditional HPLC based SEC methods
• Fully optimized column chemistry significantly reduces the requirement for high salt concentration mobile phases
• QC tested with relevant proteins, ensuring unmatched batch-to-batch consistency and increased confidence in validated QC release methods