Which scene on the ceiling shows God separating light from darkness?

The scene showing God separating light from darkness is The Separation of Light from Darkness. It represents the first day of Creation as described in the opening chapter of Genesis.

The fresco is one of nine central panels on Michelangelo’s ceiling. In the biblical sequence it comes first, before the scenes of the Creation of the Sun, Moon and Plants and the Separation of Land and Water. Michelangelo depicts God with outstretched arms, dramatically dividing brightness from shadow as he rises above the viewer.

An easily missed detail is that Michelangelo painted this first event in the Genesis narrative last among the nine central panels, probably completing it in 1512. The composition is also remarkably compact and technically demanding, using foreshortening and the ceiling-fresco technique known as sotto in su, which makes figures appear to project upward.

The scene is often confused with the Creation of the Sun and Moon, but that panel shows God creating the heavenly bodies. The Separation of Light from Darkness concerns the earlier division of day and night.