Chromatography encompasses a family of separation techniques that distribute sample components between two phases: a stationary phase (solid or liquid immobilized on a solid support) and a mobile phase (liquid, gas, or supercritical fluid). Components that interact more strongly with the stationary phase migrate more slowly, while those with greater affinity for the mobile phase elute earlier. This differential migration produces the separation.
The fundamental parameter describing retention is the retention factor (k), defined as k = (t_R − t_M) / t_M, where t_R is the retention time and t_M is the void time. The selectivity (α) between two components is the ratio of their retention factors: α = k₂/k₁. Complete separation requires both adequate selectivity and sufficient column efficiency. The resolution equation brings these together:
R_s = (√N / 4) * (α − 1 / α) * (k₂ / 1 + k₂)
where N is the number of theoretical plates. This equation shows that resolution can be improved by increasing N (longer columns, smaller particles), optimizing α (changing the stationary phase or mobile phase composition), or adjusting k (changing eluent strength).
Column efficiency is described by plate theory, which treats the column as a series of discrete equilibration steps (theoretical plates). The Van Deemter equation relates height equivalent to a theoretical plate (HETP) to linear velocity (u):
HETP = A + B/u + Cu
The A term represents eddy diffusion (packing quality), the B term represents longitudinal diffusion (significant at low flow rates in GC), and the C term represents resistance to mass transfer (important at high flow rates in HPLC). The minimum of this van Deemter curve defines the optimal mobile phase velocity for maximum efficiency.
Chromatographic methods are classified by mobile phase (liquid, gas, supercritical fluid) and by format. Column chromatography uses packed or capillary columns. Planar chromatography (thin-layer chromatography, TLC) separates on a flat adsorbent layer. Within liquid chromatography, modes include normal-phase (polar stationary phase), reversed-phase (nonpolar stationary phase), ion-exchange, size-exclusion, and affinity chromatography, each suited to particular analyte classes.
