Comparison of InVial and AboveVial mode in fraction collection on a Vanquish Analytical Purification LC system

Significance of the topic

The precise collection of HPLC fractions underpins a wide range of analytical and preparative workflows in pharmaceutical, biotechnological, and industrial settings. Ensuring minimal cross-contamination and solvent evaporation during fraction collection preserves sample integrity, supports reliable downstream analysis, and improves reproducibility in purification protocols.

Goals and overview of the study

This study compares two fraction collection modes—AboveVial and InVial—implemented on the Thermo Scientific Vanquish Analytical Purification LC system. The objectives were to evaluate:

• Cross-contamination risks and carryover between fractions
• Solvent evaporation effects during storage at elevated temperature
• Collection speed and needle positioning accuracy

Methodology and instrumentation

The Vanquish Analytical Purification system comprised a quaternary pump, split sampler CT autosampler, column compartment, diode array detector (DAD), and an integral fraction collector, all controlled by Chromeleon CDS 7.3.1. Key parameters included a C18 column (150 × 4.6 mm, 2.6 µm), 1.0 mL/min flow, 10 µL injection, and 254 nm detection. Fraction collector chamber temperature was set to 40 °C to stress evaporation effects. Five standards (uracil, acetanilide, benzophenone, hexanophenone, octanophenone) at 5 mmol/L in 80% acetonitrile were separated using a gradient from 75:25 to 5:95 (H₂O:ACN) over 8.5 min.

Main results and discussion

Repeatability tests (n = 6) showed retention time RSDs below 0.03% and peak area/height RSDs below 0.3%, confirming excellent chromatographic stability. No measurable carryover was observed for either collection mode after a 30 s needle wash.

When stored for 4 h at 40 °C, InVial-collected fractions (sealed by cap piercing, needle 3 mm above vial bottom) maintained nearly constant weight (–1.6% to –3.0%) and peak area (100.7%–101.8% of original). In contrast, AboveVial fractions (needle hovering 2 mm above vial top) showed significant solvent loss, especially for late eluting, high-organic content peaks (weight loss up to 52%; apparent concentration increases from 138.7% to 201.8%).

Benefits and practical applications

The Vanquish fraction collector offers two complementary modes:

• AboveVial mode for rapid, low-contamination transfer across a wide range of containers
• InVial mode for sealed-cap collection that minimizes evaporation even at elevated temperatures

Flexible needle height adjustment and integration with Chromeleon CDS make it suitable for high-throughput screening, purification of sensitive compounds, and automation-driven workflows.

Future trends and possibilities for use

Advances in nano- and micro-fractionation, tighter coupling with mass spectrometry, and AI-driven method optimization will further enhance the precision and throughput of preparative LC. Integration of robotics and custom scripting in Chromeleon CDS promises even greater flexibility in adaptive fraction collection strategies.

Conclusion

The Thermo Scientific Vanquish Analytical Purification LC system with AboveVial and InVial modes delivers robust, reproducible fraction collection. InVial mode effectively prevents solvent evaporation by collecting through sealed caps, while AboveVial mode offers the fastest transfer and minimal cross-contamination. Together, they address diverse purification challenges in analytical laboratories.

References

1. Koehler D.; Fabel S.; Acworth I.; Shen G. Principles of fraction collection using the Vanquish HPLC and UHPLC systems. Thermo Scientific Technical Note 72940; 2022.
2. Shi L.; Li Y.; Liu Y.; Gao L.; Wang H.; Yan Y.; Wu Y.; Ran L.; Jin Y.; Acworth I.; Koehler D.; Fabel S. Setting the start position for flexible fraction collection by using custom variables in Chromeleon CDS. Thermo Scientific Technical Note 002751; 2024.