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Motion of a liquid through a narrow space due to surface tension and contact with the walls; it draws the chromatographic mobile phase through a thin-layer chromatography coating.

The chromatographic mobile phase carries solutes through the apparatus. In liquid chromatography it is a liquid solvent or chemical solution.

The eluent is the supplied chromatographic mobile phase. Its composition can alter both solute solubility and competition for adsorption sites, so changing the eluent can change retention even with the same chromatographic stationary phase.

For a dilute solute distributed between two liquid regions at local chemical equilibrium, define the chromatographic partition coefficient asHere $c_m,c_s$ are concentrations expressed in the same units, $V_m,V_s$ specify accessible volume in the chromatographic mobile phase and immobilized liquid chromatographic stationary phase, respectively, $n$ is total solute amount of substance, and $f_m$ is the fraction of that amount of substance in the chromatographic mobile phase. Assume a constant chromatographic partition coefficient, rapid exchange, unchanged phases and temperature, and no chemical reaction. A chemical equilibrium population still exchanges molecules; it is not permanently divided into two sets.

The amount of substance balance proves the expression for $f_m$. Increasing either $K$ or $V_s/V_m$ reduces the mobile fraction, illustrating why a concentration ratio alone cannot determine retention. For surface adsorption, specify an adsorption storage law instead of assigning an arbitrary liquid volume to the solid.

An original discrete model makes the separation visible. In each identical cell, first establish local chemical equilibrium, then advance only the mobile fraction by one cell; let the stationary fraction wait. Repeat twelve times, without any additional spreading. With $V_s=V_m$, stipulated chromatographic partition coefficients $K_A=1/3$ and $K_B=3$ give $f_A=3/4$ and $f_B=1/4$. Each cycle contributes mean displacement $f_m$ cells, so the mean positions after twelve cycles are nine cells and three cells. Redistribution itself broadens each band: the cell positions have a binomial distribution with parameters $12,f_m$. These dimensionless chosen values illustrate transport, not measurements of any particular substances.

Chromatography separates components of a mixture through different migration rates in contact with a chromatographic stationary phase while a chromatographic mobile phase transports them. Liquid chromatography describes the physical state of the transporting phase; size-exclusion chromatography describes a separation mechanism. Classification by phase and classification by mechanism answer different questions. In ideal size-exclusion chromatography, differential access to liquid-filled pores replaces binding as the source of differential migration.

In many other forms of chromatography, a solute repeatedly leaves the chromatographic mobile phase and returns to it. Reversible adsorption holds it at a surface; partition distributes it between the chromatographic mobile phase and an immobilized liquid chromatographic stationary phase. Spending more time stationary means less progress with the moving fluid. Two initially overlapping components can therefore emerge as separate bands without either being permanently trapped. Chromatography is useful both for comparing a sample with reference substances and for collecting separated components; a visible band need not contain just one substance.

Relative retention belongs to the sample together with the chosen chromatographic stationary phase, chromatographic mobile phase, eluent composition, and temperature. Changing these conditions can change the separation order. Chemical polarity helps explain interactions in some systems but is not a universal ranking rule. In thin-layer chromatography, a spot's TLC retardation factor is consequently conditional evidence about identity, rather than a fixed identifier intrinsic to a molecule.

The chromatographic mobile phase supplied to a chromatography apparatus.

Thin-layer chromatography spreads a thin chromatographic stationary phase on a supporting plate. A small sample spot starts on a pencil origin line. The lower edge contacts the eluent, whose level must stay below that origin; capillary action draws the chromatographic mobile phase upward. Components exchange with the chromatographic stationary phase as the wet region advances. The leading wet edge is the solvent front. Mark it after removing the plate, before solvent evaporation erases it.^[ref]

Juniata College's Science in Motion protocol grinds vegetable tissue with about $5\mathrm{mL}$ of acetone, transfers the extract, adds about $2\mathrm{mL}$ of hexane, and specifies sampling the upper layer after separation. It spots a silica gel plate with a capillary tube, drying between repeated applications; a reference pigment occupies another lane. A closed jar contains about $0.5\mathrm{cm}$ depth of 70:30 hexane–acetone eluent, below the pencil origin. The plate is removed before the front reaches the top; the front is marked immediately. Spot centres and front are measured from the origin.^[ref]

The protocol's standard provides a comparison control. It specifies no added water in this extraction and reports no measured plate positions or temperature. Its tabulated pigment values are guidance, not observations from this run.^[ref]

A useful additional control is an eluent blank, checking contamination. Compare sample and standard on the same plate, and optionally apply both together at a third origin position. Agreement under identical conditions supports a proposed identity but cannot prove it: different substances can migrate together. A second eluent giving different selectivity strengthens the comparison. One spot cannot establish purity, and a missing visible spot can reflect poor detection rather than absence. Overloading, broad spots, or an uneven solvent front also weaken the readout. These are interpretation checks, not extra observations claimed for the protocol.