The idea of separation of powers cannot be traced back to any specific event. Scholars tend to argue that the Framers of the US Constitution got the idea of separation of powers from Montesquieu's writings, but those writings were A) not an event and B) not the first time anyone had come up with the idea of separation of powers.
In general, the Americans looked at their experience under British rule. They decided that the British Parliament did not have enough power as a legislative body because it was not sufficiently separated from the executive branch. From this experience, and from Montesquieu's writings, the Framers decided that each branch of government needed to have its own sphere of power so it would be strong enough to prevent the other branches from becoming tyrannical.
So, it was not any given event that started the idea of separation of powers. Instead it was the Framers' analysis of their whole experience under British rule.