ACQUITY UPC2 BEH, CSH, and HSS Columns - CARE AND USE MANUAL

Significance of the Topic

The advent of supercritical fluid chromatography (SFC) with ACQUITY UPC 2 BEH, CSH, and HSS columns has transformed analytical separations by delivering high efficiency, broad selectivity, and rapid analysis times. These columns leverage advanced hybrid and silica-based particle chemistries to address complex sample matrices in pharmaceutical, food, environmental, and petrochemical applications. Their robust construction and integrated eCord tracking further ensure reproducible performance and simplified lifecycle management.

Study Aims and Overview

This manual was developed to guide users through the proper care, operation, and maintenance of ACQUITY UPC 2 columns. Key objectives include:

• Describing safe handling and installation procedures for BEH, CSH, and HSS columns on ACQUITY UPC 2 systems
• Outlining initial equilibration, efficiency testing, and routine operation parameters
• Detailing troubleshooting, cleaning, regeneration, and storage protocols
• Introducing the eCord Intelligent Chip for column tracking and data management

Methodology and Instrumentation

Columns:

• Ethylene-Bridged Hybrid (BEH), Charged-Surface Hybrid (CSH™), High-Strength Silica (HSS) stationary phases
• Particle sizes: 1.7 μm and 3.5 μm; pore diameters 100–130 Å; surface areas 185–230 m2/g

Instrumentation and Setup:

1. ACQUITY UPC 2 System with CO2 delivery (≥99.97% purity) and binary modifier pump
2. VanGuard™ pre-columns for inlet protection
3. eCord® Intelligent Chip integrated into the column heater
4. High-pressure tubing and fittings sized to minimize extra-column band broadening (≤0.005" i.d.)

Operating Conditions:

• Pressure tolerance up to 18,000 psi; recommended maximum 6,000 psi
• Temperature range up to 60 °C; typical backpressure regulator set at 1,200 bar
• Mobile phases: CO2 with organic modifiers (methanol, isopropanol) and additives (TFA, ammonium formate) at ≤20 mM
• Flow rates calculated per column dimensions to avoid overpressure (e.g., 2.1 × 50 mm @ 3.5 mL/min for 100% CO2)

Main Results and Discussion

Installation and Equilibration:

• Columns shipped dry; equilibrate with ≥10 column volumes of chosen mobile phase
• Initial efficiency determined by USP plate count and tailing factor under normal-phase LC conditions

Performance Optimization:

• Sample preparation in weak solvents reduces peak distortion; filtration or centrifugation removes particulates
• Maintaining tubing internal diameter critical to band spreading; wider tubing causes significant peak broadening

Troubleshooting:

• Co-solvent pump priming issues lead to absent or delayed peaks
• Frosting at fittings indicates leaks; tighten compression screws or replace fittings after depressurization

Cleaning and Regeneration:

• Sequential flushing with polar (water, methanol, THF) or non-polar (hexane, dichloromethane) solvents as appropriate
• Aging effects mitigated by storing columns in pure CO2 to preserve retention stability
• Regeneration on UPLC systems involves rinsing with water or dilute amine solutions followed by IPA to remove residual additives

eCord Tracking:

• Stores manufacturing batch data, performance chromatogram, and user history (up to 50 sample sets)
• Automates documentation of injection counts, pressure and temperature extremes, and system suitability results

Advantages and Practical Applications

The integration of ACQUITY UPC 2 columns and eCord technology offers significant benefits:

• Enhanced method reproducibility and column-to-column consistency
• Streamlined installation with minimal extra-column volume and leak-free fittings
• Reduced downtime through clear troubleshooting and maintenance workflows
• Reliable tracking of column usage and performance over its lifecycle

These features support high-throughput laboratories in pharmaceutical QA/QC, natural product profiling, lipidomics, and chiral separations.

Future Trends and Potential Uses

Emerging directions for SFC and ACQUITY UPC 2 technology include:

• Development of novel stationary phases for challenging polar and chiral analytes
• Integration with automated sample preparation (SPE, SFE) and AI-driven method development
• Expanded use of green and bio-based modifiers to reduce environmental footprint
• Real-time column health monitoring via networked eCord analytics

Conclusion

ACQUITY UPC 2 BEH, CSH, and HSS columns, combined with the eCord Intelligent Chip and robust maintenance protocols, deliver high-performance SFC separations with reproducible results and simplified lifecycle management. Adhering to recommended installation, operation, and regeneration workflows ensures maximum column longevity and reliable analytical outcomes.

References

• Waters Corporation. ACQUITY UPC 2 BEH, CSH, and HSS Columns Care & Use Manual. Part Number 720004349EN; 2015.