Chromatography (TLC, HPLC, GC-MS)

Principle

• Chromatography separates compounds by their different distribution between a stationary phase and a moving (mobile) phase.

Thin-layer chromatography (TLC)

• Stationary phase: silica gel on a plate; mobile phase: solvent mixture moving up by capillary action.
• Sample is spotted above the solvent level; the plate is developed in a closed tank.
• Spots are visualised by UV light, iodine vapour or sprays (e.g. ninhydrin for amino acids).
• Rf = distance moved by compound ÷ distance moved by solvent front; compared with standards.
• Simple and cheap; used for drug screening and amino acid or sugar screening; semi-quantitative only.

High-performance liquid chromatography (HPLC)

• Liquid mobile phase is pumped at high pressure through a packed column.
• Modes: reverse phase (non-polar C18 stationary phase, polar mobile phase), normal phase, ion exchange, size exclusion.
• Isocratic (constant) or gradient mobile phase; detectors: UV/visible, fluorescence, electrochemical, mass spectrometry.
• Retention time identifies a compound; peak area or height quantifies it, usually against an internal standard.
• Uses: HbA1c and haemoglobin variants, catecholamines and metanephrines, vitamins, therapeutic drugs.

Gas chromatography–mass spectrometry (GC-MS)

• Volatile (or derivatised) compounds are carried by an inert gas (helium, nitrogen or hydrogen) through a heated capillary column.
• The mass spectrometer ionises separated compounds and sorts fragments by mass-to-charge ratio (m/z), producing a spectrum matched to a library.
• Regarded as a confirmatory reference method for drugs of abuse and toxicology.

Quality control and pitfalls

• Use internal standards (often isotope-labelled), calibrators and controls with every run.
• Column contamination, air bubbles, pressure changes and carryover alter retention and peaks.
• In LC-MS/MS, matrix effects (ion suppression) must be assessed.

Clinical use

• Toxicology, therapeutic drug monitoring, inborn errors of metabolism, steroid profiling, haemoglobinopathy screening.