Reverse-Phase Chromatography
Definition
Liquid chromatography with a non-polar stationary phase (most commonly octadecyl-bonded silica, called C18) and a polar mobile phase (water mixed with methanol or acetonitrile). The most common mode for small-molecule drug analysis because most analytes have enough hydrophobicity to retain on C18 and the aqueous mobile phase is cheap and LC-MS friendly.
- Stationary phase
- Non-polar, commonly C18-bonded silica
- Mobile phase
- Polar, water with methanol or acetonitrile
- Elution order
- Polar compounds elute first, non-polar last
- Common use
- Small-molecule drug analysis by LC-MS
Common questions
Why is reverse-phase preferred over normal-phase for most forensic drug analysis?+
Most drug analytes have enough hydrophobic character to retain well on C18, the aqueous-organic mobile phase is inexpensive, and it is directly compatible with mass spectrometry ionisation sources, which normal-phase organic solvents generally are not.
How does analyte polarity affect elution order in reverse-phase mode?+
More polar compounds interact weakly with the non-polar stationary phase and elute early, while non-polar or more hydrophobic compounds partition more strongly onto the C18 phase and elute later as the mobile phase's organic content increases.
What column property most affects separation of closely related drug analogues?+
Particle size and pore structure of the C18 packing affect efficiency and resolution, and gradient steepness of the organic mobile-phase ramp is the main variable analysts tune to separate structurally similar analogues such as designer drug analogues.
Related terms
- Densitometric Scanning
- The HPTLC quantitation step. After the developed plate dries, a Camag TLC Scanner 4 sweeps a narrow slit of UV or visible...
- Diode Array Detector (DAD)
- A UV-Vis detector that records the full 190 to 800 nm absorbance spectrum of every peak as it elutes, instead of a...
- Isocratic vs Gradient Elution
- Isocratic means the mobile-phase composition stays constant through the run; simple, reproducible, but limited resolving power for wide-polarity mixtures. Gradient means the...
- Method Validation Parameters
- The numbers a forensic method must hit before it is considered fit for routine use: linearity R² > 0.999 over the working...
- Sub-2 µM Particle Column
- A UHPLC column packed with 1.6 to 1.8 µm silica particles instead of the conventional 5 µm. Smaller particles give sharper peaks...