This one! Ten and Rose, after going through so much together and being so connected, they get separated. If they hadn't ended up in different universes, they probably would have had a thing, but then that would have ruined the series altogether so I'm glad it worked out that way. At the same time, I cried. He had to drop her into the different universe to save her life, but then he couldn't reach her. She had faith though, and after years and years and years of searching, she finally finds a holographic projection of him in Bad Wolf Bay. Definitely the most heartbreaking separation of The Doctor and a companion.
Favorite incredibly heartjerking line from this episode: "I'm burning up a sun just to say Goodbye."