High-Throughput Gradient Optimization by Easily Minimizing Delay Volume

Importance of the Topic

High‐throughput gradient HPLC is critical in analytical chemistry for reducing analysis time, increasing sample throughput, and lowering solvent consumption. Techniques that shorten column re‐equilibration and minimize gradient delay volume enable rapid separations while preserving chromatographic performance.

Study Objectives and Overview

This application note evaluates a straightforward pump modification and column selection strategy to optimize gradient delay volume and re‐equilibration in Agilent Rapid Resolution High Throughput (RRHT) systems. The goal is to demonstrate how reduced system and column volumes improve gradient response, peak shape, and overall analysis speed.

Used Instrumentation

• Agilent 1200 RRLC system
• G1365C multiwavelength detector with 3 mm path, 2 µL micro flow cell, 254 nm
• G1312B binary pump SL configured for standard or low delay volume
• ZORBAX RRHT columns: Eclipse Plus C18 (2.1×30 mm, 1.8 µm) and Eclipse XDB‐C8 (4.6×30 mm, 1.8 µm)

Methodology

The mobile phases comprised 0.1 % formic acid in water (A) and in acetonitrile (B). Sample mixtures contained uracil and alkyl phenones (C3–C14). Gradient profiles ramped from 70 % to 100 % B in 0.5 min at 1.5 mL/min for narrow‐bore and 5 mL/min for standard‐bore columns. The G1312B pump was reconfigured by rerouting capillaries to reduce internal volume from ~600–800 µL to ~120 µL. A 200‐µL static mixer and firmware update were optionally added to suppress baseline disturbances in highly aqueous gradients.

Main Results and Discussion

• Column re‐equilibration time for a 4.6×150 mm analytical column (~7.5 min) was reduced to ~1.5 min using 2.1×30 mm RRHT columns, representing a 5× decrease in time.
• Low delay volume pump configuration sharply reduced isocratic lag, sharpening later‐eluting peaks and delivering a 29 % faster cycle on narrow‐bore and 14 % on standard‐bore columns.
• Substituting a C8 phase further shortened retention, decreasing analysis time from ~0.9 min to ~0.6 min for alkyl phenones.
• Baseline oscillations induced by differential UV absorption in aqueous gradients were eliminated by installing a 200‐µL static mixer and applying a firmware update optimized for low‐volume operation.

Benefits and Practical Applications

• Significant gains in sample throughput with minimal changes to existing methods.
• Reduced solvent usage and operating costs due to shorter re‐equilibration and gradient delays.
• Enhanced flexibility for method scaling and transfer between column formats.
• Improved peak shapes and resolution in gradient analyses of small molecules.

Future Trends and Possibilities

• Integration of automated flow‐path switching to dynamically adjust delay volume per method requirements.
• Development of microfluidic or on‐column mixing technologies to further reduce system volume and improve gradient fidelity.
• Expansion of RRHT stationary phase chemistries for customized selectivity at high throughput.
• Coupling low‐delay configurations with mass spectrometry for rapid LC–MS workflows in QA/QC and discovery screening.

Conclusion

Minimizing system delay volume and leveraging short RRHT columns delivers pronounced improvements in HPLC gradient throughput. Simple pump rerouting and optional static mixing mitigate delay‐induced artifacts, while stationary phase selection offers further speed gains. These strategies enhance productivity, reproducibility, and cost efficiency in modern analytical laboratories.

Reference

1. Optimizing Performance of the Agilent 1200 Rapid Resolution LC System, Agilent Publication G1312-90300, March 2006.