Agilent 1260 Infinity II Flexible Pump with Blend Assist

Importance of the Topic

Efficient optimization of buffer and modifier concentrations in liquid chromatography is essential for achieving high resolution, reproducibility, and throughput in pharmaceutical, proteomic, and quality control laboratories. The ability to adjust mobile-phase composition online accelerates method development and reduces manual intervention, improving consistency and resource utilization.

Objectives and Study Overview

This technical overview evaluates the Agilent 1260 Infinity II Flexible Pump enhanced with Blend Assist in two key scenarios:

• Optimization of trifluoroacetic acid (TFA) concentration for the separation of a 10-peptide standard.
• A multimethod sequence analyzing three distinct compound groups—sulfa drugs, a testosterone mixture, and a peptide standard—by varying ammonium formate concentration without manual solvent exchange.

Methodology and Instrumentation

High-purity LC-grade solvents and stock solutions of TFA or ammonium formate were prepared. Blend Assist was configured to dilute stock solutions online, creating targeted modifier concentrations in ternary or quaternary gradient runs. Key chromatographic parameters were:

• Columns: AdvanceBio Peptide Map (2.1×100 mm, 2.7 µm) and ZORBAX StableBond C18 (2.1×50 mm, 1.8 µm).
• Flow rates: 0.5–1.0 mL/min; column temperatures: 30–60 °C; injection volumes: 1–5 µL.
• DAD detection at relevant UV wavelengths (220–254 nm) with reference filtering at 360 nm.

Used Instrumentation

• Agilent 1260 Infinity II Flexible Pump (G7104C) with Blend Assist and V380 Jet Weaver Mixer.
• Agilent 1260 Infinity II Vialsampler (G7129C) with integrated cooler.
• Agilent 1260 Infinity II MCT (G7116A) column compartment.
• Agilent 1260 Infinity II Diode Array Detector HS (G7117C) with Max-Light cartridge cell.

Key Results and Discussion

In peptide separations, increasing TFA from 0.05% to 0.3% improved resolution of critical peak pairs (Neurotensin/Angiotensin I) from coelution to baseline separation (resolution rising from 0.99 to 2.25). Comparison of Blend Assist versus manually mixed 0.3% TFA mobile phases showed near-identical retention times and resolution, with retention time RSDs <0.02% using Blend Assist and <0.01% manually.

In the multimethod sequence, Blend Assist enabled in-line adjustment of ammonium formate:

• Sulfa drugs at 200 mM achieved RT RSDs <0.10%.
• Testosterone CIII and acetate at 50 mM showed RSDs <0.12%.
• Sigma peptide mix at 25 mM yielded RSDs well below the 0.15% specification.

These results confirm excellent precision, reproducibility, and flexibility across diverse analyte classes without manual solvent exchanges.

Advantages and Practical Applications

Blend Assist streamlines mobile-phase optimization by:

• Eliminating time-consuming manual preparation of multiple modifier concentrations.
• Maintaining high accuracy and precision in gradient formation.
• Facilitating complex multimethod sequences for diverse compound sets in a single run.

This approach benefits pharmaceutical development, proteomics, and routine QA/QC by reducing solvent waste and operator workload.

Future Trends and Opportunities

Potential advancements include integration of real-time feedback algorithms for dynamic gradient adjustment, application to multidimensional chromatography workflows, and coupling with machine learning models for predictive method optimization. Extending Blend Assist to other analytical platforms could further accelerate automated method development.

Conclusion

The Agilent 1260 Infinity II Flexible Pump with Blend Assist offers a robust solution for rapid, precise online variation of buffer and modifier concentrations. Demonstrated across peptide and small-molecule separations, it ensures high resolution and retention time reproducibility while simplifying multimethod workflows.

Reference

1. Huesgen A.G. Fast and Flexible Optimization of Modifier Concentrations Using an Agilent 1290 Infinity LC System with Blend Assist, Agilent Technologies Technical Overview, publication number 5991-2169EN, 2013.