Thermo Scientific μPAC HPLC Column robustness in bottom-up proteomics

Significance of the topic

Robust and long-living LC columns are critical for uninterrupted bottom-up proteomics workflows. Column failures lead to system downtime, altered retention times and peak shapes, and increased maintenance efforts.

Objectives and study overview

This study evaluates the operational lifetime and resilience of Thermo Scientific μPAC micro pillar array columns under standard bottom-up proteomics conditions and challenging sample types. A single 200 cm μPAC column was monitored over six months and 3526 injections, including HeLa digest, blanks, Cytochrome C references, and three sets of difficult samples.

Methodology and instrumentation

The experiments used a Thermo Scientific 200 cm μPAC column operated at 1 μL/min with a 1–50 % B gradient over 30 min (A=H2O+0.1 % TFA or FA, B=ACN+0.1 % TFA or FA), column temperature 35 °C and UV detection at 214 nm. The μPAC column features a microfabricated array of 2.5 μm-spaced silicon pillars providing high permeability, low dispersion and low backpressure.

Main results and discussion

Retention time of six reference peptides remained highly stable (CV<2 %) across 1000 HeLa injections. Peak width, asymmetry and column backpressure (235 bar at 1 μL/min) showed negligible drift. Three challenging sample sets containing Triton X-114, NP-40 detergents and particulate precipitates were injected without observable loss of chromatographic performance or clogging. Stability metrics were confirmed by intermediate Cytochrome C reference runs.

Benefits and practical applications

μPAC columns offer extended operational lifetime, reduced downtime and consistent chromatographic performance even with contaminant-rich samples. Their low backpressure permits long columns for high resolution and sharp peaks, benefiting proteomics laboratories, QA/QC and industrial analytics.

Future trends and potential applications

Advancements in microfabrication may yield even longer and more efficient pillar array columns. Integration with high-throughput and automated platforms, broader applications in metabolomics and clinical proteomics, and coupling with advanced MS instrumentation are promising directions.

Conclusion

The μPAC micro pillar array column demonstrated exceptional robustness over six months and 3526 injections, maintaining stable retention times, peak shapes and backpressure even with challenging samples. This performance supports reliable, high-resolution proteomics analyses.

Reference

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