WHAT IT IS
Nano-Liquid Chromatography (Nano-LC) is an ultra-low-flow format of liquid chromatography that operates with column internal diameters below 100 µm and flow rates in the range of 50–1000 nL/min. By miniaturizing the separation system, Nano-LC maximizes sensitivity, especially when coupled with mass spectrometry (MS). This makes it particularly valuable in proteomics, metabolomics, and biomarker discovery, where sample amounts are limited and trace-level detection is required. Compared with standard HPLC and micro-LC, Nano-LC provides the highest ionization efficiency for electrospray MS, enabling the detection of low-abundance analytes in complex biological matrices.
HOW IT WORKS
Nano-LC uses the same principles as conventional LC but with further downscaled hardware and flow paths:
Mobile Phase Delivery - Specialized nano-flow pumps deliver extremely low flow rates (nL/min) with high stability. Flow splitting from standard pumps is sometimes used but may reduce precision.
Sample Introduction - Autosamplers or manual injection systems are adapted for nanoliter to microliter volumes. Careful design prevents dead volume, which can distort peaks at such low flows.
Column Separation - Columns have very narrow internal diameters (20–100 µm), typically packed with sub-2 µm particles or monolithic stationary phases. These dimensions improve separation efficiency but increase sensitivity to clogging.
Detection - Nano-LC is almost always coupled to mass spectrometry. The extremely low flow enhances electrospray ionization efficiency, resulting in strong signals even for low-concentration analytes.
Solvent Use - Solvent consumption is minimal, often less than a milliliter per day, lowering costs and waste.
ADVANTAGES
Maximum Sensitivity: Ultra-low flow rates enhance electrospray efficiency, providing strong MS signals from minute sample amounts.
Minimal Sample Requirement: Enables analysis of scarce biological material, such as single-cell proteomics or rare clinical samples.
Low Solvent Consumption: Uses extremely small amounts of solvent, reducing operating costs and waste.
High Resolution: Narrow columns and small particles yield sharp, efficient separations.
Essential for Proteomics: Widely regarded as the gold standard for peptide and protein LC-MS workflows.
APPLICATION AREAS
Proteomics: Identification and quantification of peptides and proteins at trace levels.
Metabolomics: Detection of low-abundance metabolites in complex biological systems.
Clinical Biomarker Discovery: Analysis of limited patient samples for diagnostic research.
Pharmaceutical Development: Characterization of biopharmaceuticals and trace impurities.
Single-Cell and Micro-Sample Analysis: Enables molecular profiling when sample volume is extremely limited.
CHALLENGES AND LIMITATIONS
System Complexity: Requires specialized nano-flow pumps, fittings, and low-dead-volume connections.
Column Fragility: Very narrow columns are easily clogged and must be handled carefully with filtered, clean samples.
Reproducibility Issues: Nano-LC is more sensitive to minor variations in flow, temperature, and pressure than micro- or standard LC.
Detector Dependence: Works best with mass spectrometry; optical detectors (UV, fluorescence) are less effective at such low volumes.
Method Transfer Difficulty: Scaling methods between nano-LC and larger formats requires substantial adjustment of flow rates, gradients, and injection volumes.