Pushing the Boundaries of Chromatographic Separation with Inert HPLC Column Hardware

Importance of the Topic

The analysis of biologically relevant molecules such as acidic metabolites and phosphorylated peptides is critical in many fields including metabolomics proteomics and pharmaceutical research.
However interactions with metal surfaces in columns and flow paths can lead to adsorption signal loss and poor peak shapes which compromises sensitivity and quantitation.
The development of inert column hardware aims to overcome these limitations by minimizing interactions between analytes and metal surfaces thus improving analytical performance.

Objectives and Study Overview

This study evaluates the performance stability inertness and analytical sensitivity of a new HPLC column platform featuring Ultra Inert coated stainless steel columns.
Key goals include comparing chromatographic separation quality signal recovery and durability against standard stainless steel hardware across diverse analyte classes under various conditions.

Methodology and Instrumentation

• Samples synthetic phosphopeptides acidic metabolites and nucleotides
• Columns Ultra Inert HILIC and C18 columns and standard stainless steel alternatives and competitor inert columns
• Instrumentation Agilent 1290 Infinity II bio LC system Agilent 6545XT AdvanceBio LC Q TOF mass spectrometer and Agilent 1290 Infinity Flexible Cube for stress tests
• Mobile phases formic acid water acetonitrile buffer systems and ion pairing reagents
• Stress tests exposure to strong acid base concentrated PBS and elevated temperature ion pairing conditions over six weeks

Key Results and Discussion

• Chromatographic performance Ultra Inert columns showed up to threefold higher signal intensities sharper peaks and reduced tailing factors for peptides nucleotides and acidic metabolites compared to stainless steel columns
• Chemical stability coated columns maintained signal integrity after prolonged exposure to harsh solvents and high temperature demonstrating minimal performance loss
• Reproducibility four production lots of coated columns delivered consistent signal recovery highlighting reliable batch to batch manufacturing
• Phosphopeptide detection Ultra Inert C18 columns outperformed standard and competitor inert columns yielding higher peak areas and improved sensitivity at low injection amounts
• System synergy combining Ultra Inert columns with a bio inert LC system produced maximum signal gains while replacing only the column in an existing stainless steel LC still resulted in significant sensitivity improvements

Benefits and Practical Applications of the Method

• Enhanced sensitivity and quantitation in metabolomics proteomics and pharmaceutical analyses
• Reduced method development time by minimizing metal related artifacts
• Extended column lifetime and consistent performance under demanding workflows
• Compatibility with existing LC platforms enabling easy upgrade to inert hardware

Future Trends and Potential Applications

• Integration with high throughput bioanalysis and single cell omics workflows
• Expansion of inert coatings to microfluidic systems and capillary platforms
• Application in analysis of complex biomolecules such as phospholipids glycopeptides and metal containing pharmaceuticals
• Development of next generation inert stationary phases for ultra high pressure applications

Conclusion

The Ultra Inert HPLC column hardware demonstrates superior inertness stability and reproducibility leading to significant improvements in analyte recovery and chromatographic quality.
These advances address key challenges in analyzing metal sensitive biomolecules and provide a robust solution for laboratories seeking higher sensitivity and more reliable results.
By combining inert column hardware with compatible LC systems users can achieve optimal performance across a wide range of applications.

References

• Wei et al The use of HILIC Zwitterionic Phase Superficially Porous Particles for Metabolomics Analysis LCGC Supplement 2018 36(6) 30 35
• Hsiao et al Improved LC MS Methods for the Analysis of Metal Sensitive Analytes Using Medronic Acid as a Mobile Phase Additive Anal Chem 2018 90(15) 9457 9464
• Hsiao et al Troubleshooting LC Separations of Biomolecules Part 1 Background and the Meaning of Inertness LCGC Europe 2020 33(3) 122 126
• Fleitz et al Enhanced Detection of Multiply Phosphorylated Peptides and Identification of Their Sites of Modification Anal Chem 2013 85(18) 8566 8576