HANDS-ON HPLC TROUBLESHOOTING AND MAINTENANCE

Some previous knowledge of HPLC and basic experience are recommended but not essential.

The Troubleshooting Process

• Introduction to Troubleshooting
  • Define the Problem: How to accurately define the issue by gathering all relevant information and symptoms
  • Propose Hypotheses: Developing potential causes for the problem based on available data
  • Isolate the Cause: Step-by-step method to systematically isolate the root cause, such as changing one variable at a time
  • Investigate and Test: How to conduct targeted tests to verify hypotheses and confirm the issue
  • Resolve the Issue: Implement solutions, verify they worked, and monitor performance over time
• Benchmarking and Performance Qualification
  • Defining system benchmarks and how to compare against expected performance
  • Performing system qualification to ensure proper functionality after resolution
• Preventative Maintenance
  • Overview of preventive maintenance practices to avoid recurring issues
  • How to set up regular maintenance schedules for key components

Common Issues in Instrumentation

• Precision
  • Identifying issues with precision and reproducibility (e.g., high %RSD in repeated injections)
• Extra Peaks and Contamination
  • Identifying contamination sources: Mobile phase impurities, column bleed, or sample carryover
  • Cleaning columns, flushing the system, replacing solvents
• Sensitivity Issues
  • Troubleshooting loss of sensitivity: Checking detector performance, flow cell, and solvent degassing
  • Identifying causes like dirty optics, low light intensity in detectors, or improper method setup
• Method-Related Issues
  • Analysing method parameters: Flow rate, gradient, temperature, and sample preparation
  • Identifying potential method-related problems that affect performance
• Analyst Error
  • Recognising common human errors in sample preparation, instrument operation, or data analysis
  • Best practices to minimise errors through standard operating procedures (SOPs) and checklists

The Column

• Hardware
  • How the column designed with efficiency in mind: ZDV unions and connections, different port depths. Packing
• Measuring Performance
  • How to tell if a column is experiencing issues and how to diagnose specific issues using Efficiency, Asymmetry. Looking at improvements via solid core and UHPLC
• Common Issues
  • Based on technical enquiries we look at common issues such as back pressure, voids, reversing flow, resolution issues
• Cleaning and Regeneration
  • Typical column cleaning and regeneration program to maximise column life span

Instrumentation Components and Troubleshooting

• Solvent Bottles and Lines
  • Issues with solvent lines (e.g., blockages, air bubbles) and how to troubleshoot flow inconsistencies
  • Tips for maintaining solvent purity and avoiding contamination
• Degassing
  • The importance of solvent degassing and how degassing issues can lead to inconsistent performance
  • Best practices for degassing solvents (e.g., degasser units, sonication, helium sparging)
• Pumps
  • Common pump issues: Flow rate inconsistencies, pressure fluctuations, and leaks
  • Troubleshooting steps for checking seals, pistons, and valves
• Autosampler
  • Issues with sample carryover, incorrect volumes, or needle problems
  • Troubleshooting autosampler mechanics: Needle alignment, wash solvents, and rotor seals
• UV Detector
  • Common issues in UV detectors: Baseline noise, drift, or low sensitivity
  • Troubleshooting optical alignment, flow cell contamination, and lamp issues

Practical Maintenance

• Maintenance Planning
  • How to create a maintenance schedule based on instrument usage
  • Understanding the difference between routine maintenance and emergency repairs
  • Using built-in software tools for real-time diagnostics and performance monitoring
• Pump Maintenance
  • Pistons: Cleaning and replacing worn pistons
  • Seals: Checking for leaks, replacing seals, and understanding when they need replacement
  • Check Valves: Diagnosing valve blockages or failures, and how to clean/replace them
  • Seal Wash System: Ensuring seal wash system is functioning properly to avoid solvent crystallization around seals
• Autosampler Maintenance
  • Needle: Cleaning or replacing clogged needles to avoid sample volume inaccuracies
  • Needle Seat: Inspecting and maintaining the needle seat for proper sample uptake
  • Rotor Seal: Troubleshooting and replacing worn rotor seals to avoid leaks or incorrect sample injections
• Detector Maintenance
  • Cleaning the flow cell, checking UV lamp intensity, and replacing the lamp when necessary
• Diagnostic tools
  • Pressure testing: use of blanking nut to test if connections in the system can hold the system pressure without leaking
  • Detector testing: running a series of diagnostic tests such as wavelength calibration, intensity test, darkness test to ensure proper detector function
  • EMF: Setting up and monitoring EMF functions for preventative maintenance