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Yes - it's the first moment that the audience can be sure that Claudius is in fact guilty, and that the ghost (even if not the spirit of Hamlet's father) is honest. Until then, nothing has been certain.
O, my offence is rank, it smells to heaven;
It hath the primal eldest curse upon't,
A brother's murder! Pray can I not,
Though inclination be as sharp as will;
My stronger guilt defeats my strong intent,
And, like a man to double business bound,
I stand in pause where I shall first begin,
And both neglect. What if this cursed hand
Were thicker than itself with brother's blood,
Is there not rain enough in the sweet heavens
To wash it white as snow?
No ambiguity there! Hope it helps!
Claudius is in a small prayer chamber where he has gone after watching "The Murder of Gonzago". It was common for king's to have their own private prayer rooms so that they could pray in private. Hamlet, knowing the location of the king's prayer room, also knows a way in which to watch him pray. In many stage adaptations, the chamber also has a confessional box for the priest and that is where Hamlet goes to overhear Claudius' confession.
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