Increase sensitivity in UHPLC-MS analysis by minimizing post-column dispersion

Importance of Minimizing Post-Column Dispersion in UHPLC-MS

Minimizing extra-column band broadening is crucial for enhancing sensitivity, resolution, and throughput in fast-gradient UHPLC-MS, especially when analyzing complex samples requiring high peak capacity and minimal peak overlap.

Objectives and Study Overview

This study demonstrates a straightforward and cost-effective approach to reduce post-column dispersion by replacing standard capillaries with narrower internal diameter nanoViper fittings in a Thermo Scientific Vanquish Horizon UHPLC system coupled to an ISQ EM single quadrupole mass spectrometer. The method was evaluated using a mixture of nine pesticide standards under a 5-minute gradient separation.

Methodology and Instrumentation

The separation employed an Accucore Vanquish C18+ column (2.1×100 mm, 1.5 µm) with solvents A (5 mM ammonium formate 0.1% formic acid in water) and B (95:5 methanol:water containing the same additives), at 0.4 mL/min flow rate, 40 °C column temperature, and 0.1 µL injection volume. Post-column fluidics were optimized in two steps:

• Replacement of the 100 µm×550 mm Viper MP35N capillary with a 75 µm×550 mm nanoViper fitting
• Bypassing additional PEEK tubing by directly connecting the column outlet to the MS inlet and grounding union using 75 µm nanoViper fittings (550 mm and 250 mm), reducing extra fluidic paths

Used Instrumentation

• Vanquish Horizon UHPLC system (System Base, Binary Pump H, Split Sampler HT, Column Compartment with active preheater)
• Thermo Scientific ISQ EM Single Quadrupole MS
• nanoViper and Viper MP35N Fingertight capillary fittings

Key Results and Discussion

• Replacing the 100 µm capillary with 75 µm reduced peak width by an average of 9% and increased peak height by 8%
• Full fluidic optimization yielded average improvements of 29% narrower peaks, 42% higher signal intensity, and a 36% increase in peak capacity
• The optimized setup operated at ~1250 bar, within the Vanquish Horizon’s 1500 bar limit, enabling high-pressure fast gradient separations without flow reduction

Benefits and Practical Applications

Reduced post-column dispersion enhances detection sensitivity and chromatographic resolution in high-throughput analyses, such as pesticide screening, by delivering sharper peaks and higher signal-to-noise ratios within short run times.

Future Trends and Applications

• Extension of low-dispersion strategies to other mass spectrometry platforms and analyte classes
• Integration with high-resolution MS and microflow UHPLC for further sensitivity gains
• Automated fluidic optimization in commercial UHPLC–MS workflows

Conclusion

Targeted reduction of extra-column volume through narrower capillary fittings in UHPLC-MS significantly improves chromatographic performance and detection sensitivity in fast-gradient methods. The robust pressure capability of modern UHPLC systems supports this optimization without sacrificing throughput.

References

1. Thermo Scientific. Vanquish – Binary Pump H Product Specifications 71186. Thermo Fisher Scientific; 2021.
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3. Zhou Z, De Pra M, Steiner F, Desmet G, Eeltink S. Assessing effects of ultra-high-pressure liquid chromatography instrument configuration on dispersion, system pressure, and retention. J Chromatogr A. 2020;1634:461660.
4. Wang X, Stoll DR, Schellinger AP, Carr PW. Peak capacity optimization of peptide separations in reversed-phase gradient elution chromatography: Fixed column format. Anal Chem. 2006;78:3406–3416.