Demonstrating Inertness for the Analysis of Nucleotides on the Agilent 1290 Infinity II Bio LC

Significance of the Topic

The inertness of a liquid chromatography system is essential for accurate analysis of metal-sensitive biomolecules such as nucleotides. Unwanted interactions between phosphate groups and metal surfaces can cause peak tailing, peak loss and poor reproducibility, undermining data quality in pharmaceutical, biochemical and environmental applications.

Objectives and Study Overview

This application note evaluates the inertness of the Agilent 1290 Infinity II Bio LC using a test mixture of adenosine (neutral control) and a nonhydrolyzable adenosine diphosphate analogue (AMPcP). By comparing peak area ratios for five consecutive injections, the study quantifies potential adsorption of phosphate-containing analytes to metal surfaces.

Methodology and Instrumentation

The system comprised:

• Agilent 1290 Infinity II Bio Flexible Pump (G7131A)
• Agilent 1290 Infinity II Bio Multisampler with sample thermostat (G7137A, option 101)
• Agilent 1290 Infinity II Multicolumn Thermostat with biocompatible heat exchanger (G7116B, G7116-60071)
• Agilent 1290 Infinity II Variable Wavelength Detector with biocompatible 2 µL flow cell (G7114B, G1314-60189)
• ACQUITY PREMIER HSS T3 column, 1.8 µm, 2.1×50 mm

Chromatographic conditions used a gradient from 5% to 95% acetonitrile with 10 mM ammonium acetate and 0.1% trifluoroacetic acid, at 0.4 mL/min, 35 °C, detection at 260 nm. The sample contained 7.6 nmol AMPcP and 8 nmol adenosine in ultrapure water.

Main Results and Discussion

Overlay of five injections showed highly consistent retention times (RSD < 0.1%) and peak areas (RSD < 0.5%). The AMPcP/adenosine area ratio averaged 0.95±0.01, indicating minimal adsorption of the phosphorylated analogue. These metrics confirm the system’s ability to handle iron-sensitive analytes without signal loss or distortion.

Benefits and Practical Applications

• Reliable quantification of nucleotides and other phosphate-bearing molecules
• Reduced peak tailing and improved peak shape for biochromatography
• High confidence in method reproducibility for QA/QC and research labs
• Compatibility with high salt, extreme pH and strong chaotropes without degradation

Future Trends and Applications

Emerging applications include analysis of phosphorylated peptides, glycan profiling and metal-binding small molecules. Further expansion may involve automation for high-throughput screening and integration with mass spectrometry for detailed structure elucidation.

Conclusion

The Agilent 1290 Infinity II Bio LC demonstrates outstanding inertness for nucleotide analysis, yielding consistent retention and area reproducibility while minimizing adsorption of phosphate groups. This low-adsorption platform enhances confidence in biochromatographic results and supports diverse analytical workflows.

References

1. Wakamatsu A et al. Mass Spectrometry Applications in Separation Sciences 2005, 28(14), 1823–1830.
2. Fekete S. LCGC Europe 2021, 34(6), 245–248.
3. Schneider S. Agilent Technologies application note, 5994–4392EN, 2021.
4. Patel A et al. Waters Corporation application note, 720007105EN, 2021.