Getting started with μPAC Neo HPLC columns

Importance of the Topic

High‐resolution, high‐throughput nanoLC–MS proteomic analyses demand robust and reproducible chromatographic separations with minimal backpressure. Maskless micro‐pillar array technology (µPAC) has revolutionized nanoLC separations by replacing packed beds with highly ordered pillar arrays. The second‐generation µPAC Neo columns improve resolution, sensitivity, and throughput for deep proteome profiling of scarce biological samples.

Objectives and Overview

This guide details the installation, conditioning, and best practices for Thermo Scientific™ µPAC™ Neo HPLC columns on the Thermo Scientific™ Vanquish™ Neo UHPLC system. It compares µPAC Neo to first‐generation µPAC and conventional packed‐bed columns, outlines two workflows—direct injection and trap‐and‐elute—and presents strategies for gradient and flow‐rate optimization to maximize chromatographic performance and MS utilization.

Methodology and Instrumentation

µPAC Neo columns feature a monolithic microfluidic pillar array embedded in an aluminum casing with integrated grounding and nanoViper™ capillary connections. Three analytical formats are described:

• 50 cm nonporous “low‐load” column (75 µm ID) for sub‐nanogram samples.
• 50 cm superficially porous column for 0.01–0.5 µg loads.
• 110 cm superficially porous column for up to 2 µg, single‐shot analyses.

Two workflows were evaluated:

• Direct injection: sample is loaded directly onto the analytical column.
• Trap‐and‐elute: sample is first captured on a trapping column (C8 or C18 chemistry) before elution onto the µPAC Neo analytical column.

Key instrumentation:

• Vanquish Neo UHPLC system in nano/capillary mode
• Thermo Scientific nanoViper™ capillaries (20 µm ID)
• Thermo Scientific EASY‐Spray™ bullet emitter with integrated liquid junction
• Thermo Scientific µPAC Neo and trapping columns
• Thermo Scientific Proteome Discoverer™ software with Sequest HT and Chimerys search algorithms

Main Results and Discussion

µPAC Neo columns deliver 15–25 % narrower peak widths (FWHM) and higher chromatographic resolution compared to first‐generation µPAC. Pressure–flow profiles show operation up to 750 nL/min at <450 bar, facilitating rapid separations with reduced backpressure.
Direct injection of 200 ng HeLa digest on the 50 cm column yielded ~4 600 protein groups in 67 min gradients. Conditioning with 0.1 % TFA minimized hydrophilic peptide breakthrough; manual loading volumes ≤1.5 µL improved peak shapes and identifications.
Trap‐and‐elute workflows increased MS utilization by up to 95 %, as loading and equilibration occur in parallel. Using a C18 µPAC trapping column, 50 ng HeLa digest produced ~4 400 proteins in 30 min separation windows, with ~14 000 peptide‐spectrum matches.
Flow‐rate optimization combining fast loading at 750 nL/min with analytical elution at 250 nL/min boosted sensitivity and depth of coverage. For example, reducing flow to 250 nL/min after 2 min increased proteome identifications by over 20 % in 20 min gradients.

Benefits and Practical Applications

µPAC Neo columns offer:

• Enhanced resolution and sensitivity for limited or complex samples
• Lower backpressure vs packed beds, enabling longer columns and steeper gradients
• Reproducible, low‐carryover separations due to nonporous and superficially porous pillars
• Flexible workflows—direct injection for maximum depth, trap‐and‐elute for high throughput and cleaner separations
• Efficient equilibration and high MS utilization via fast loading/column equilibration modes

Applications include single‐cell proteomics, biomarker discovery, QA/QC in biopharmaceutical analysis, and high‐throughput screening.

Future Trends and Possibilities

Advances may include further miniaturization for true single‐cell sensitivity, integration with ion mobility (e.g., FAIMS), and on‐the‐fly gradient and flow‐rate adjustments driven by real‐time MS feedback. Emerging fabrication techniques could yield even higher order pillar geometries and chemistries for specialized separations, enabling multi‐omics and clinical applications at scale.

Conclusion

The µPAC Neo platform on the Vanquish Neo UHPLC system delivers robust, reproducible nanoLC separations with unmatched resolution, sensitivity, and throughput. By following optimized installation, conditioning, and method development protocols—tailoring column format, workflow, loading volume, solvent composition, and flow rate—laboratories can achieve deeper proteome coverage and higher productivity for modern proteomic challenges.

References

1. Stejskal K, de Beeck JO, Durnberger G, Jacobs P, Mechtler K. Ultrasensitive NanoLC-MS of Subnanogram Protein Samples Using Second Generation Micropillar Array LC Technology with Orbitrap Exploris 480 and FAIMS PRO. Anal Chem. 2021;93(25):9134–42.
2. Zheng R, Arrey TN, Hakimi A, et al. Fast, Sensitive, and Reproducible Nano- and Capillary-Flow LC-MS Methods for High-throughput Proteome Profiling Using the Vanquish Neo UHPLC System Hyphenated with the Orbitrap Exploris 480 MS. Thermo Fisher Scientific Technical Note TN000138. 2022.
3. Stejskal K, op de Beeck J, Matzinger M, Dürnberger G, Boychenko A, Jacobs P, Mechtler K. Deep Proteome Profiling with Reduced Carryover Using Superficially Porous Microfabricated NanoLC Columns. Anal Chem. 2022;94(14):5421–28.