Chapter 59, when Ayane tearfully realizes that, unlike Sawako and Chizuru, she has never really liked or dated anyone because of love.
Chizuru flat out refused to accept Ryu's feeling in his second confession.
Chizuru's reaction to finding about Ryu's feelings for her, at which point she believes that their friendship will never return to the way it was.
Chizu: *crying* He's not my brother anymore.
Chapter 63, which has the flashback of Ryu's family grieving over the death of his mother.
Even in that chapter, little Chizuru making onigiri (like his mother made) and bringing them to Ryu, which finally cracks his stoic shell to show his grief, stands out as a tear-jerking scene. Also notable (although it's a small thing) is Chizu tightening his scarf, something his mother always used to do for him.
The chapter in itself is sad enough, but seeing Ryu, of all people, break down and cry is the real kicker.
At the end of chapter 66, Sawako starts to cry and delivers this line. It's not so much the line itself as how upset she looks when she says it.
Sawako: I wasn't the one who created the distance between us. He was.
Chapter 82: Just when Chizu seems to be slowly accepting that she might have feelings for Ryu, he announces that he's going away to study at university. Made worse by the fact that she's not been planning on going anywhere, and a large reason for this seems to have been the fact that she thought he'd be staying, too.
And later, in Chapter 88: "Since when did that start? Since when was I the only one who noticed him?"
Ayane's Character Development, whilst astonishingly good, hits very hard at points. It's sad to realise that the group's Cool Big Sis is actually unhappier and more insecure than anyone realised.