c. 1400, separacioun, "a severing, detaching, cutting apart, act of removing or disconnecting one thing from another," from Old French separacion (Modern French séparation) and directly from Latin separationem (nominative separatio) noun of action from past-participle stem of separare "to pull apart," from se- "apart" (see secret (n.)) + parare "make ready, prepare" (from PIE root *pere- (1) "to produce, procure"). Alternative separateness (1650s) tends to hold to the meaning "distinct character or state, fact of being separate."
The specific sense of "sundering of a married couple, limited divorce" (without dissolution of the marriage tie) is attested from c. 1600. Sense in printing in reference to proportionate monochrome representations of a color photograph is from 1922.
Separation of powers is attested by 1792, from French séparée de la puissance (Montesquieu, 1748). The idea was discussed in several places in "The Federalist" (1788), but not in that exact phrase (e.g. separation of the departments of power, No. 81). In psychology, the child's separation anxiety is attested from 1943.