Overcoming Barriers with UHPLC to Achieve Maximum Performance

Importance of the Topic

Ultra-high-performance liquid chromatography (UHPLC) offers unmatched separation efficiency and throughput in modern analytical laboratories. However, system inefficiencies such as excessive extra column and delay volumes can negate these advantages, leading to broader peaks, lower resolution, and longer run times. Understanding how to minimize these barriers is essential for routine applications in pharmaceutical development, food safety testing, environmental monitoring, and quality control.

Objectives and Study Overview

This work examines strategies to achieve maximum UHPLC performance by addressing two primary challenges: extra column volume and system delay volume. Using a series of test compounds (alkylphenones, aromatic acids, and catechins), the authors compare a default Agilent 1290 Infinity LC configuration with an optimized low-dispersion setup. The goal is to quantify the impact of hardware modifications and software features on chromatographic parameters.

Methodology and Instrumentation

The study employs an Agilent 1290 Infinity LC system configured in two modes:

• Default configuration: extra column volume ≈ 11 µL, system delay ≈ 127 µL.
• Optimized low-dispersion setup: extra column volume ≈ 4 µL, reduced delay using ADVR software.

Key components:

• Column: Agilent ZORBAX RRHD Eclipse Plus C18, 2.1 × 50 mm, 1.8 µm.
• Ultra Low Dispersion kit and Max Light Cartridge Flow Cell.
• Capillaries: 0.075 mm i.d. tubing, shortest possible lengths between modules.
• Autosampler modifications: bypass mode after injection via ADVR to minimize delay.
• Detection: UV at 210–280 nm and MS (Agilent 6410A QQQ, ESI+).

Analytical conditions vary by sample type, typically employing water/acetonitrile gradients (0.2–0.6 mL/min), column temperatures of 26–40 °C, and injection volumes of 1–4 µL.

Main Results and Discussion

Comparison of chromatographic performance between default and optimized systems revealed:

• Reduction in extra column volume from 11 µL to 4 µL sharpened peak widths (PW1/2 decreased from ~0.021 min to ~0.012 min for alkylphenones) and improved resolution (Rs from ~2.96 to ~3.67).
• Minimized delay volume led to earlier elution of early-retained compounds and more accurate gradient delivery, especially for fast gradients on small columns.
• Proper capillary installation eliminated mixing chambers and voids that cause tailing; misaligned ferrules were identified as a common source of peak distortion.
• Data acquisition rates for UV and MS detections were optimized to balance peak shape, height, and signal-to-noise ratio; excessively fast rates increased noise, while slow rates broadened peaks.

Benefits and Practical Applications

Optimizing UHPLC hardware and software offers:

• Shorter analysis times and increased sample throughput.
• Higher resolution and sensitivity for complex mixtures.
• Reduced solvent consumption and operating costs.
• Robust performance in QA/QC laboratories and regulated environments.

Future Trends and Opportunities

Ongoing developments are expected to further enhance UHPLC performance:

• Advanced low-dispersion fittings and microfluidic front ends.
• Integration of AI-driven system diagnostics and adaptive gradient control.
• Novel stationary phases designed for ultra-fast separations.
• Green chemistry approaches with aqueous and bio-based solvents.

Conclusion

Effective minimization of extra column and delay volumes, combined with proper capillary installation and optimized data acquisition, unlocks the full potential of UHPLC systems. Implementing low-dispersion hardware and software tools such as ADVR ensures faster, sharper, and more reproducible separations critical to modern analytical workflows.

Reference

1. Mack A, Long W, Link J, Joseph M. Optimizing Performance of an Agilent 1290 Infinity LC System for Ultra Low Dispersion. Agilent Publication 5990-9502EN, April 2012.
2. Mack A. Improve a Waters Method for Aromatic Acids by Using an Agilent ZORBAX RRHD Column and an Agilent 1290 Infinity LC. Agilent Publication 5991-1681EN, December 2012.
3. Mack A. Fully Using Agilent High Efficiency Columns with LC/MS. Agilent Publication 5990-8623EN, January 2012.
4. Agilent Posters: Tips for Optimizing UHPLC Sensitivity and Throughput. Agilent Publications 5991-1749EN and 5990-7594EN, January 2013/March 2011.