Each metabolite is drawn from the mobile phase into the LC column. Nonpolar analytes bind weakly to the stationary phase and elute quickly; polar analytes bind strongly and elute late — this is the retention factor k. At the column exit, the electrospray source ionizes molecules with an efficiency that depends on the ion mode and the analyte's acidity, and the mass spectrometer records an m/z peak that is scored against a reference library.
retention time: t_R = L / v, v = v0 · F / (1 + k)
retention factor: k = P_col · affinity · 6
ESI+ efficiency: η = 1 − acidity (detected if η > 0.25)
ESI− efficiency: η = acidity
ID score: s = exp( −((mz_i − mz_db) / (0.01·mz_i))² )
identified if max(s) ≥ threshold
- New Sample — injects a fresh batch of 48 metabolites (known + unknown) into the column.
- Mobile Phase Flow — scales elution velocity v, shortening every retention time t_R.
- Column Polarity — raises the retention factor k for polar analytes, spreading peaks apart (chromatographic resolution).
- ID Confidence Threshold — the minimum Gaussian mass-match score s required to call a detected peak "identified".
- Ionization Mode — switches which acidity range of molecules ionizes efficiently in the ESI source.
This mirrors a real untargeted LC-MS/GC-MS metabolomics pipeline: chromatographic separation, ESI ionization, peak picking, and library-matched compound identification feeding downstream statistics.