For an item to separate between another element of the same type, and another surface: Rosh HaShanah 27b; Succah 37b [2x]
Distinguishing where [s]he doesn't care about the presence of the separation, under ordinary circumstances: Shabbat 114a; Eruvin 4b; Pesachim 45a-b; Succah 6b
Distinguishing where the majority of a surface is not covered: Eruvin 4b; Succah 6b
Considering an item which is added to a Mitzvah-item solely for beauty, to be a separation when one is supposed to hold the actual item: Succah 37a
Whether to distinguish between a separation on one side, or both sides, of a garment/saddle-blanket: Shabbat 114a
If an item is considered important for the prohibition against Leaven on Pesach, that makes it count for Separation, during Pesach, to prevent Impurity of Vessels: Pesachim 46a
Considering hair to be a separation, as a Law spoken to Moshe at Sinai: Eruvin 4b; Succah 5b, 6a-b
Honey/Ink/Milk/Blood, when Liquid, as separations: Shabbat 120b; Pesachim 65b; Zevachim 35a
Reeds: Shabbat 120b
Fat on clothing: Shabbat 114a
Fat on a saddle-blanket: Shabbat 114a
Separation of a type with its own type: Yoma 58a
Separation of one type to another type: Yoma 58a
Separations during Immersion
The disqualification of immersion due to a separation on the skin: Eruvin 4b, Succah 6a
If an item is considered important for the prohibition against Leaven on Pesach, that makes it count for Separation, during Pesach, to invalidate Immersion of Vessels: Pesachim 46a