Tips and Tricks of Faster LC Analysis Without Capital Investment

Significance of the topic

High-performance liquid chromatography (HPLC) is a workhorse of analytical laboratories, but long run times, high solvent consumption and instrument backlog limit throughput. This collection of practical strategies demonstrates how to accelerate LC analyses by 2–10× with minimal or no capital investment, improving sample throughput, reducing waste disposal costs and optimizing existing hardware and personnel resources.

Objectives and study overview

The primary goal is to present easy-to-implement tips and tricks that enable faster isocratic and gradient separations through column and instrument adjustments. The tutorial covers:

• Speed gains achievable by particle size and column length optimization
• Flow-rate, temperature and gradient time adjustments
• Maintaining efficiency (N), resolution (Rs) and selectivity during acceleration
• Using Agilent Method Translator for seamless method transfer to rapid resolution LC (RRLC)

Methodology and instrumentation

This approach relies on four key elements:

• Column optimization: moving from 5 μm → 3.5 μm → 1.8 μm particles and shortening columns (150 mm → 50–30 mm) to maintain plate count with reduced run times
• Maintaining constant linear velocity: adjusting flow rates proportionally when changing column inner diameter to preserve chromatographic performance
• Gradient optimization: reducing gradient time in proportion to column volume (Vm) and/or increasing flow to keep gradient steepness (k*) constant
• Temperature elevation: raising column temperature (up to 90 °C) to lower mobile phase viscosity, enhance mass transfer kinetics and sharpen peaks

Instrumentation examples use an Agilent 1100 HPLC series upgraded to the 1200 SL RRLC system, including:

• μ-Degasser and binary pump SL capable of >400 bar
• High-pressure autosampler and column compartment SL
• VWD/DAD detector with variable data rates (up to 80 Hz) for narrow peaks

Main results and discussion

Key findings include:

• 2–3× time reduction: halving column length and using smaller particles cut isocratic runs from ~12 min to ~5 min without loss of resolution
• 3–5× speed gains: 1/3 column length with 1.8 μm packing delivers sub-2 min separations at ambient pressure limits
• 10–20× acceleration: combining short 50 mm columns, high flow (2–5 mL/min) and elevated temperatures produces sub-1 min gradient assays, albeit requiring >400 bar capability
• Resolution and sensitivity: smaller particles boost efficiency by up to 160%, resolution by 60% and signal-to-noise ratios remain comparable despite faster runs
• Software support: Agilent Method Translator automates injection volume, flow, gradient and detector transfer, ensuring robust conversion from conventional to RRLC methods

Benefits and practical applications of the method

Implementing these strategies offers laboratories:

• Increased daily sample throughput and more efficient “home by five” schedules
• Reduced mobile phase consumption (up to 90% savings) and waste disposal costs
• Enhanced utilization of existing LC modules without immediate investment in new hardware
• Improved chromatographic performance for QA/QC, pharmaceutical impurity profiling and high-speed screening workflows

Future trends and applications

Emerging directions include:

• Wider adoption of sub-2 μm and monolithic columns for ultrafast separations
• Integration of high-pressure (>1000 bar) systems for sub-minute assays
• Micro- and nano-bore formats with low solvent consumption and enhanced MS sensitivity
• Automated method translation tools and AI-driven optimization algorithms for rapid method development
• Advanced detector technologies with higher sampling rates for peak profiling in extreme high-speed LC

Conclusion

Substantial speed improvements in HPLC can be realized through a combination of column, flow, temperature and software adjustments without major capital outlay. By leveraging smaller particles, shorter columns, proportionally adjusted gradients and existing instrument capabilities, laboratories can achieve dramatic reductions in analysis time while preserving or enhancing resolution and sensitivity.

References

No specific literature references were provided in the source document.