In the episode "Chuck Versus the Alma Mater", after Chuck finds out that Bryce Larkin was protecting Chuck all along and that he never actually betrayed him, we get a flashback at the end, which shows Chuck and Bryce meeting for the first time, this was pretty heartbreaking, considering that Chuck thought Bryce was dead at that point.
The episode "Chuck Versus the Marlin". Because of those damn FULCRUM agents, the government decides that Chuck will be taken from his job, family, and friends, and basically stuck in a basement for the foreseeable future. The episode itself is heartbreaking from the get-go, but it hits you square in the gut when Chuck is waiting to be taken away and tries his best to face his fate and is clearly failing. One particularly touching moment was when Chuck said the upside to having his life taken from him, was that maybe perhaps Sarah could visit him in his cell, and maybe see how they really felt about each other.
The fountain conversation in "Chuck Versus the Breakup".
"Chuck Versus the Pink Slip":
[[spoiler:Sarah: It's not that simple. You don't know who you're working for. It's complicated. Nothing is real. This... (clasps Chuck's hand) this is simple. This is a real life. We have to go, Chuck. This is it. Are you coming? Chuck: (hands Sarah back the train ticket) I can't. I'm sorry.]]
...which makes Chuck's speech in "Chuck Versus the Three Words" all the more poignant:
"Look, Sarah, I know... I know that you're probably very hurt... you're probably hurt that I didn't run away with you in Prague. I did that... I'm sorry. You have to know that you were everything that I ever wanted. But how could do that... how could I run away with you... how could I be with you knowing what I'd turn my back on... you know, knowing what I had in my head could help a lot of people? And you're the one that taught me that being a spy is about choosing something bigger... it's about putting aside your own personal feelings for the greater good, and that's what I chose. I chose to be a spy for my friends and my family and you. I chose to be a spy because... Sarah, I love you."
In "Chuck Versus the Beard", Morgan gets fed up with Chuck being so secretive that he de-friends Chuck. The fact that at this time Chuck was so weighed down by how lonely and conflicted he was that he couldn't even flash properly made an already upsetting moment so much worse.
"Chuck Versus the Tic Tac" is just one punch in the gut after another, all of them involving Casey and all of them hurting about as much as an actual punch in the gut from Casey would. Not enough to find out that "John Casey" is really "Alexander Coburn," a soldier who faked his death and took on a new identity in order to serve his country? Try seeing yet another of his mentors going rogue and forcing him into committing treason to save the life of his former fiancée, who still thinks he's dead. And then meeting the daughter he didn't know he had. And just because this episode hasn't been cruel enough to Casey already, it ends with him being dismissed from service. If you never thought Adam Baldwin could break your heart, think again.
"Chuck Versus the Subway": Shaw murders Chuck's father right in front of him, with Ellie watching the whole thing from her hiding place. Just so that Chuck wouldn't be able to use his Intersect-given fighting skills.
"Chuck Versus Phase Three" had Sarah, after tearing apart much of the jungles of Thailand, finding Chuck nearly lobotomized. In tears, she finally tells Chuck what she needed to tell him:
"Chuck, I want to spend the rest of my life with you. I don't care if you have the Intersect or not. Without you, I'm nobody. I'm nothing but a spy. Come back to me, Chuck. I want to marry you."
The beginning of "Chuck Versus the Cliffhanger" when Sarah is rushed to hospital after being poisoned by Vivian:
Sarah:(delirious) Chuck? Chuck: Hey. Hey, baby, hey, I'm right here. Sarah: You look worried. Chuck: No, you know, just... it's just wedding stuff. Sarah:Don't freak out. We're ready for it. (Her eyes close) Chuck: Sarah... Baby, I'm going to fix this. We're going to get married and be together. Forever. I love you.
The last several minutes to "Chuck Versus The Bullet Train" were absolutely gutwrenching.
To elaborate Sarah, who was suffering the effects of the Intersect (namely agonizing headaches and memory defects), goes after Big Bad Quinn who's being pursued by Chuck and Casey. She confronts him in the back car of the train only to be tranquilized by Quinn. Chuck watches helplessly as Quinn separates the car from the train, taking off with his wife, just as they were about to quit the spy business and start a life together.
It Gets WorseSarah is taken to a warehouse where Quinn completely wipes away her mind, making her forget everything, including Chuck. Keep in mind, that it's as terrible as it sounds, with Sarah suffering physical and mental torture as this is happening. Sadly, Chuck and Casey track her to the warehouse, but arrive too late.
Think that's bad? The succeeding episode, "Chuck Versus Sarah" (and no, it's not a metaphorical title, so...) is far worse, especially the last half of the episode. To put it simply, if you think Zachary Levi couldn't make you weep buckets? Think again.
To further elaborate, The last half has Chuck taking Evil!Sarah to their dream house, and desperately trying to restore her memories, as he tearfully tells Sarah about the great times they've had together over the past five years.
"Chuck Versus The Goodbye The whole thing and especially the final scene. However, this Troper chooses to believe the Word of God that after the final fadeout Sarah did remember everything eventually and fell in love with Chuck all over again.
This Troper really wants to believe Word of God because Chuck is an unbearably tragic series if they're not right, but isn't sure they really thought this through. Even if Sarah eventually gets everything back, she may not necessarily be the same person she was before. For this version of Sarah: Bryce and Graham's deaths are still fresh for her, she's pulled a gun on her sister-in-law, she's savagely beaten her husband and come within a hairsbreadth of killing him/Morgan/Casey, and several of the people in her support network -- Ellie, Awesome, Mama Bartowski, and Casey -- have now moved away. Even if she gets all of her memories back, and the series hints that she's moving in that direction, that's still a pretty horrible set of experiences for any person to endure, and it's likely to change her in ways that are difficult to predict.