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For thin layer or paper chromatography, this can be a very important step, especially if one or more of the components in the mobile phase have very high vapor pressures (or low boiling points).

Without the equilibration, as the mobile phase starts to move up the plate, the more volatile components will evaporate into the air space above the mobile phase faster than the less volatile ones. This is change the composition of the mobile phase (not much, but sometimes enough to change the Rf's of the unknown spots and standards).

If the mobile phase consists of only water or perhaps high boiling solvents, the equilibration step is not as important. If the mobile phase contains Methylene chloride, Diethyl ether or similar solvents, then the equilibration step can be very important in terms of reproducibility.

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