Advances in liquid chromatography–mass spectrometry (LC-MS/MS) are enabling more sensitive, cost-efficient, and sustainable workflows for oligonucleotide characterization. At ASMS 2025, Sergio Guazzotti, Biopharma and Low Flow Business Development Manager for Europe at Phenomenex, presented a low-flow micro-LC approach that achieves high-quality sequencing data while minimizing sample use, reducing exposure to toxic reagents, and lowering per-analysis costs.
The Case for Low-Flow Separation
Reducing LC flow rates has a direct impact on sensitivity. Narrower column diameters and slower flow reduce sample dilution, improving electrospray ionization (ESI) efficiency. “By using these new columns, you can inject 25 times less sample volume and still get even better signal than with a 2.1 mm analytical column,” notes Guazzotti. For example, in sequence confirmation experiments, a 2.1 mm column at 25 µL injection might yield ~80% confirmation, while the low-flow setup can achieve 98% confirmation with just 1 µL.
The new columns operate in the 2.5–5 µL/min range—faster and simpler than nano-flow LC but with much of the same approach to sensitivity gain. This design offers a practical middle ground: high performance without the handling complexity and fragility associated with sub-100 µm ID nano columns.
Comprehensive Oligonucleotide Analysis in One Workflow
The workflow supports simultaneous characterization of:
- Poly(A) tail length
- 5′ cap efficiency
- Sequence integrity
- Impurity profiles
Such integrated analysis reduces the number of runs needed, streamlining method development and QC. The columns use a core-shell stationary phase, which maintains chromatographic efficiency at reduced solvent and modifier concentrations.
Lower Cost and Higher Safety
Reverse-phase LC separations of oligonucleotides often require ion-pairing reagents alongside alcohol modifiers such as hexafluoro-isopropanol (HFIP). HFIP is costly, toxic, and—when degraded—could potentially form per- and polyfluoroalkyl substances (PFAS). Lowering both the reagent concentration and the total flow volume decreases operator exposure, environmental impact, and consumable costs.
“With low-flow micro-LC, we spend less money, expose users to less reagent, and potentially generate less PFAS,” explains Guazzotti. This sustainability angle aligns with increasing regulatory and institutional pressure to reduce hazardous chemical use in analytical laboratories.
Column and Hardware Design for Low-Flow Robustness
The low-flow columns feature a fused-silica body to eliminate bulk metal contact, paired with proprietary non-metal frit technology. This minimizes unwanted oligonucleotide adsorption, a common issue with stainless-steel frits. Maximum on-column loading is around 16 µg, which is suitable for the targeted sample sizes in this application.
To further improve usability, Phenomenex has developed integrated trap cartridges compatible with torque-limiting connection tools. These ensure consistent fittings—avoiding both under- and over-tightening—which can otherwise compromise performance or damage valve ports.
Practical Impact
The combination of higher sensitivity, reduced sample and reagent use, and simplified hardware handling makes low-flow micro-LC an attractive option for labs working with precious or expensive oligonucleotide samples. While Guazzotti does not expect every user to switch to low-flow, interest is growing among researchers seeking better data quality with lower operational costs and improved safety profiles.
Peer-reviewed publications are already available from early adopters, offering detailed method parameters for those looking to evaluate the technology in their own labs.