Improving Method Transfer by Adjustment of Gradient Delay

Importance of the Topic

In many laboratories and production sites, high-performance liquid chromatography (HPLC) methods developed on one system must be transferred to multiple instruments to maintain throughput and compliance. Variations in internal system volume and pump characteristics lead to gradient delay and shifts in retention time, undermining reproducibility and method compatibility. Addressing these discrepancies is essential for reliable quantitative and qualitative analysis across different HPLC platforms.

Objectives and Overview

This report aims to clarify the impact of system volume–induced gradient delay on chromatographic separation, demonstrate practical examples of method transfer for alkylphenones and UV absorbents (sunscreen mixture), and introduce Shimadzu’s Nexera-i i-Series LC system with the Analytical Conditions Transfer and Optimization (ACTO) function for streamlined method adjustment.

Methodology and Gradient Delay Concept

• System Volume and Gradient Delay: Defined as the volume between eluent mixing point and column inlet; larger volumes delay actual gradient start, altering retention times and resolution.
• Alkylphenone Analysis: Two systems with volumes of 1155 µL and 505 µL were compared using reversed-phase HPLC; an appropriate initial hold time (650 µL equivalent) was added to correct delay.
• UV Absorbent (Sunscreen) Analysis: Method transferred from a vendor system to Shimadzu Nexera-i MT; gradient start time was adjusted by 340 µL using ACTO to align retention times across systems.

Instrumentation

• Shimadzu Nexera-i i-Series integrated LC system featuring optimized low- and high-pressure gradient paths
• LabSolutions software with ACTO function for automatic adjustment of gradient start time
• Shim-pack VP-ODS and XR-ODS II columns, photodiode array detectors

Main Results and Discussion

In the alkylphenone study, adjusting the initial isocratic hold to compensate for a 650 µL volume difference produced virtually identical chromatograms on both systems. The sunscreen mixture transfer showed retention time errors reduced from over 1% to near zero after a 340 µL gradient delay correction via ACTO. These examples confirm that simple gradient start time adjustments can overcome inter-system variability without modifying the core gradient program.

Benefits and Practical Applications

• Ensures consistent chromatographic performance across multiple HPLC instruments
• Maintains regulatory compliance by avoiding changes to prescribed gradient profiles
• Saves development time by eliminating the need for extensive method revalidation

Future Trends and Opportunities

Continued advancement in LC-MS integration and ultrahigh-speed separations will require even finer control of system volumes and pump dynamics. Automated method transfer using software tools like ACTO may evolve to include machine-learning models for predictive adjustment and broader support for various chromatographic platforms.

Conclusion

Gradient delay caused by system volume differences is a primary source of retention shifts during HPLC method transfer. Implementing initial hold time or gradient start time adjustments offers a straightforward and regulation-friendly solution. Shimadzu’s i-Series LC systems together with the ACTO function enable efficient, reproducible method transfer across diverse HPLC platforms.

References

1. United States Pharmacopeia. General Chapter on Method Transfer and Dwell Volume Adjustments. 2016.