Peptide separation on μPAC HPLC columns

Importance of Peptide Separation

Peptide separation plays a pivotal role in proteomics by enabling detailed analysis and quality assessment of protein digests. High-resolution separations facilitate accurate component detection and are essential for validating liquid chromatography performance prior to mass spectrometry workflows.

Objectives and Study Overview

This study evaluates the chromatographic performance gains achieved using Thermo Scientific μPAC HPLC columns compared to conventional packed bed columns. A cytochrome c tryptic digest serves as a model sample to benchmark separation resolution, peak capacity, and operational versatility under varying gradient times and flow rates.

Methodology and Instrumentation

• Sample: 0.5 μM cytochrome c tryptic digest.
• Columns: μPAC pillar array (200 cm length, 18 μm pillars) and packed bed C18 (75 μm × 15 cm, 2 μm, 100 Å).
• Gradients: 1–50 % organic (solvent B) over 60 to 300 min.
• Flow rates: 300, 600, and 900 nL/min.
• Detection: UV at 214 nm.

Main Results and Discussion

• The μPAC column delivered narrow average peak widths (4σ ≈ 0.22 min) and symmetry factors near 1.2.
• Compared to packed bed, μPAC reduced peak broadening by ~19 % for gradients longer than 120 min.
• High permeability allowed operation up to 900 nL/min at moderate pressures (<300 bar), whereas packed bed columns faced pressure limitations.
• Peak capacity increased by up to 25 % on μPAC columns, with peak capacity per unit time improved by up to 14 % at 300 nL/min and up to 25 % at higher flow rates.

Practical Benefits and Applications

The enhanced resolution and throughput make μPAC columns attractive for proteomics workflows, quality control of HPLC systems, and complex peptide separations prior to mass spectrometry. Their stable performance across extended gradients supports in-depth analysis of low-abundance peptides.

Future Trends and Opportunities

Advancements in pillar array designs may enable even longer columns and higher flow rates, further boosting peak capacity and reducing analysis time. Integration with next-generation mass spectrometers and automation platforms could streamline proteomic analyses and expand applications in clinical and industrial settings.

Conclusion

Thermo Scientific μPAC HPLC columns offer a versatile solution for high-resolution peptide separations, combining narrow peak widths, elevated peak capacity, and robustness over a broad range of flow rates. They represent a significant upgrade over traditional packed bed columns for advanced proteomics and analytical chemistry applications.

Used Instrumentation

• μPAC HPLC column (200 cm, 18 μm pillar array).
• Packed bed C18 column (75 μm × 15 cm, 2 μm, 100 Å).
• Nanoflow liquid chromatography system capable of 100–1000 nL/min.
• UV detector at 214 nm.

References

• De Malsche W, Gardeniers H, Desmet G. Experimental Study of Porous Silicon Shell Pillars under Retentive Conditions. Anal. Chem. 2008;80:5391–5400.
• De Malsche W, Op De Beeck J, De Bruyne S, Gardeniers H, Desmet G. Realization of 1 × 10^6 Theoretical Plates in Liquid Chromatography Using Very Long Pillar Array Columns. Anal. Chem. 2012;84:1214–1219.