Correlation Between the Adsorption of Acidic Analytes on Stainless Steel Columns and Their Ionic Charge

Significance of the Topic

The interaction between acidic analytes and stainless steel surfaces can compromise HPLC results by reducing signal intensity and peak quality. Understanding and mitigating these interactions is essential for reliable analysis of nucleic acids, peptides, proteins, glycans and acidic small molecules in pharmaceuticals, biochemistry and environmental monitoring.

Study Objectives and Overview

This application note investigates how the ionic charge of acidic compounds influences their adsorption on stainless-steel HPLC columns. By comparing conventional hardware to MaxPeak Premier inert-surface technology, the study seeks to correlate analyte charge with observed peak area losses.

Methodology and Instrumentation

• Sample Preparation
  Stock solutions at 1 mg/mL; working solutions at 50 µg/mL in water.
• Chromatography
  System: ACQUITY Premier BSM with PDA detection at 210 nm
  Columns: ACQUITY Premier and standard stainless-steel CSH Phenyl-Hexyl, 2.1×50 mm, 1.8 µm
  Mobile phases: 0.1% formic acid in water (A) and methanol (B)
  Gradient: 100% A (2.00 min) to 95% B over 6.00 min, hold 1.15 min, re-equilibrate (total 10.33 min)
  Flow rate: 0.5 mL/min; Column temp 30 °C; Sample temp 10 °C.
• Mass Spectrometry
  MS: Xevo TQ-S micro in negative ESI mode
  Capillary 1.5 kV; Cone 20 V; Desolvation 350 °C, 650 L/h
• Data Handling
  MassLynx V4.1 for acquisition and calculation of peak area ratios.

Main Results and Discussion

All tested acidic compounds (nine analytes) exhibited lower peak areas on stainless steel compared to inert-surface columns. Peak area ratios (SS/MP) were below 1 and decreased with more negative ionic charge and lower mass load. A strong correlation (R2≈0.92) was observed at the lowest load between calculated analyte charge at pH 3.0 and adsorption severity. Differences among acidic moieties (sulfonic, phosphonic, carboxylic) highlighted the influence of functional group count and acidity on metal interaction.

Benefits and Practical Applications

• Inert-surface columns reduce acidic analyte loss, improving sensitivity and peak shape.
• Predictive correlation with analyte charge enables method development guidance.
• Applicable to pharmaceutical quality control, metabolomics, and biomolecule analysis.

Future Trends and Potential Applications

Advancements may include expanded datasets for nonlinear charge–adsorption modeling, tailored surface chemistries for specific analyte classes and integration with predictive software or machine learning to streamline method selection in complex matrices.

Conclusion

The study demonstrates that analyte ionic charge is a key predictor of adsorption on stainless-steel HPLC hardware. MaxPeak Premier inert surfaces effectively mitigate these losses, offering reliable analysis of acidic compounds across diverse applications.

References

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