Single Source Solution for Low Flow Chromatography

Significance of topic

Low-flow electrospray ionization mass spectrometry (ESI-MS) has become essential for applications demanding enhanced sensitivity and efficient sample utilization. Achieving reliable performance across nanoflow and microflow regimes addresses the diverse needs of proteomics, bioanalysis, and small molecule quantitation. A single, versatile ion source simplifies workflow transitions, optimizes throughput, and maintains robust operation under varying analytical demands.

Study objectives and overview

This whitepaper presents the development and characterization of the OptiFlow Turbo V Source, a unified solution for low-flow chromatography. The goals were to:

• Design a plug-and-play source supporting 100–1000 nL/min nanoflow and 1–200 µL/min microflow.
• Ensure consistent sensitivity comparable to dedicated nanoflow sources.
• Maintain robustness and rapid regime switching with minimal user tuning.

Methodology and instrumentation

Sample preparation employed the SWATH® Acquisition Performance Kit for nanoflow and signature peptides or small molecule mixtures for microflow and analytical flow tests. Chromatography systems included the NanoLC™ 425 with nano and low-microflow modules and the M5 MicroLC for microflow experiments. Mass spectrometers were the QTRAP® 5500/6500/6500+ and the TripleTOF® 6600+, each equipped with the OptiFlow interface. Key components were SteadySpray Probes and Electrodes: 20 µm ID for 100–1000 nL/min, 25 µm and 50 µm for 1–50 µL/min, and 50 µm for 50–200 µL/min. Data processing used MultiQuant™ for targeted MRM, PeakView® for SWATH Acquisition, and ProteinPilot™ for DDA protein identification.

Key findings and discussion

• Design optimization via Design of Experiments minimized positional tuning and fixed optimal electrode protrusion and gas flows, creating a broad “sweet spot” for sensitivity.
• Microflow tests at 3 µL/min showed long-term robustness over 1150 injections with <4% CV in peptide MRM ratios and similar performance in positive/negative polarity switching over 1050 runs (~18 days).
• Sensitivity comparisons between 500 µL/min and 3 µL/min revealed 4.8–6.7-fold average area gains and 8.4–33.5-fold signal-to-noise improvements for small molecules.
• Nanoflow configuration yielded stable operation over 30 days without electrode clogging, and proteomics benchmarking on the TripleTOF 6600+ delivered protein and peptide IDs within ±2% of a fully optimized NanoSpray® III source.

Benefits and practical applications

• Rapid flow regime switching in minutes enables optimal sensitivity or throughput per experiment.
• Pre-optimized SteadySpray Probes eliminate manual tuning, reducing setup time.
• Broad compatibility with high-throughput bioanalysis, sensitive proteomics, and small molecule quantitation across polarity modes.

Future trends and potential applications

Integration of single-source, low-flow platforms aligns with trends toward multi-omic workflows and high-throughput screening in drug discovery. Further miniaturization, automated probe exchange, and real-time source diagnostics promise enhanced usability and reproducibility in clinical, environmental, and industrial analytics.

Conclusion

The OptiFlow Turbo V Source successfully combines nanoflow sensitivity with microflow robustness in a single, user-friendly interface. Pre-configured probes and electrodes deliver consistent performance, minimize downtime, and enable flexible method development across a wide range of applications.

References

1. Covey TR, Schneider BB, Javaheri H, Ivosev G, Corr JJ, Kovarik P. Electrospray and MALDI mass spectrometry: Fundamentals, instrumentation, practicalities, and biological applications. In: ESI, APCI, and MALDI. Wiley; 2010:443–490.
2. Schneider BB, Javaheri H, Covey TR. Ion sampling efficiency under conditions of total solvent consumption. Rapid Commun Mass Spectrom. 2006;20:1538–1544.
3. SWATH Acquisition Performance Kit Standard Operating Protocol.
4. OptiFlow Interface for TripleTOF 6600 System. SCIEX Technical Note RUO-MKT-02-7219-B.