ACQUITY UPLC and ACQUITY PREMIER Peptide BEH C18, 130 Å and 300 Å Column

Importance of the Topic

The precise separation of peptides is a cornerstone of modern proteomics, biopharmaceutical development, and quality control laboratories. High-efficiency reversed-phase chromatography enables accurate peptide mapping, impurity profiling, and rapid method development. Waters’ ACQUITY UPLC and ACQUITY PREMIER Peptide BEH C18 columns are engineered to deliver reproducible performance, narrow batch-to-batch variability, and seamless integration with advanced instrumentation.

Study Objectives and Overview

This care and use manual is designed to guide analytical scientists through every phase of column usage—from unpacking a new column to long-term storage. It covers:

• Initial installation and equilibration procedures
• Routine performance verification and efficiency testing
• Recommended operational parameters for pH, temperature, pressure, and solvent systems
• Cleaning, regeneration, and storage protocols
• Implementation of eCord™ Intelligent Chip technology for column tracking
• Representative chromatographic conditions for peptide separations

Methodology and Instrumentation

Columns are packed with BEH (bridged ethyl hybrid) C18 particles available in 130 Å and 300 Å pore sizes, manufactured under cGMP/ISO 9002 conditions. Each column is tested with acidic, basic, and neutral probes, and a peptide digest for quality control. Key procedural steps include:

• Connector installation using gold-plated tubing and compression fittings
• Column equilibration with at least ten column volumes of mobile phase
• Performance verification via theoretical plate and tailing factor determination
• Sample preparation guidelines to ensure compatibility and prevent particulate fouling

Used Instrumentation

• Waters ACQUITY UPLC System or ACQUITY PREMIER System
• Peptide BEH C18 Columns (2.1 × 50, 100 or 150 mm lengths; 130 Å and 300 Å pore sizes)
• Column heater module with eCord™ Intelligent Chip reader
• Empower™ and MassLynx™ Software for method control and data capture

Main Results and Discussion

A representative separation of a tryptic digest of cytochrome c on a 2.1 × 100 mm Peptide BEH C18 column at 35 °C and 0.20 mL/min demonstrates baseline resolution of 19 peptide fragments in a 26 min gradient (0–36% acetonitrile with 0.045% TFA). Critical performance metrics such as plate count, capacity factor, and tailing factor are provided via the eCord chip. The chromatogram confirms high peak efficiency, consistent retention times, and minimal secondary interactions following column conditioning.

Benefits and Practical Applications of the Method

These columns deliver:

• High reproducibility across batches and systems for robust method transfer
• Wide pH tolerance (1–12) and temperature range (20–55 °C) for versatile applications
• Rapid equilibration and minimized carry-over with MaxPeak™ High Performance Surfaces
• Integrated eCord tracking to monitor cumulative usage, system suitability results, and maintenance history

Future Trends and Applications

As proteomic and biopharmaceutical workflows evolve, future developments may include:

• Advanced surface chemistries to further suppress non-specific binding
• Enhanced digital tracking and predictive maintenance via AI integration
• Ultra-high throughput formats and microfluidic coupling for single-cell proteomics
• Automated method optimization workflows driven by machine learning models

Conclusion

Waters’ ACQUITY UPLC and PREMIER Peptide BEH C18 columns, combined with eCord Intelligent Chip technology, offer a comprehensive solution for high-performance peptide separations. Adhering to the recommended installation, equilibration, and maintenance protocols ensures consistent results, long column lifetimes, and streamlined laboratory operations.