Stationary phase: Water molecules trapped within the cellulose matrix of chromatography paper. This is the KEY fact — the stationary phase is water, NOT the paper itself.

Mobile phase: Organic solvent (or solvent mixture) that moves up the paper by capillary action.

Technique: Spot sample near the bottom of paper → lower edge dips into mobile phase (but spots above solvent level) → solvent rises by capillary action → components separate based on their partition between water (stationary) and organic solvent (mobile).

Ascending chromatography: Solvent moves upward (most common). Descending: solvent moves downward (faster for long runs).

Detection: Amino acids visualized by spraying with ninhydrin (purple spots, except proline → yellow). Sugars with aniline-phthalate spray.

Rf (retardation factor) = distance traveled by substance / distance traveled by solvent front. Values range 0 to 1. More polar compounds have lower Rf (prefer water phase, move less). Rf is characteristic for a compound under specific conditions (temperature, solvent system, paper type) — used for identification.

Two-dimensional paper chromatography: Run in one solvent, rotate 90 degrees, run in different solvent → better separation of complex mixtures (amino acid analysis).