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One must separate challah from dough once the dough has been rolled and kneading is complete. The Mishnah (3:1) taught that one is allowed to snack from that dough prior to that point. We have also learnt that if one attempts to separate challah from flour, the separation is ineffective. The Mishnah however teaches that one can (or should) separate challah as soon as the water has been added. We shall try to understand this ruling.
The Bartenura explains that even though the main mitzvah is to separate challah from fully formed dough, the Chachamim instituted this practice to best ensure that the separated challah is tahor.
The Mishnah however adds a condition and there are two versions of the Mishnah. The first is that it is "as long as there is not five quarter kav". According to this version, there is a concern that there is not a volume of flour remaining that has not yet mixed with the water. The reason is that the separated challah would satisfy the requirement for the remaining flour, which in turn would require challah be separated from it once it mixes with the water. According to the second version, the condition is that "as long as there is five quarter kav". According to this reading, the requirement is that when separating the challah early, the flour that has mixed with the challah must be a volume that requires the separation of challah.
The Bartenura continues by citing the Yerushalmi, that one can stipulate that they are separating challah now for all the dough, sourdough and remaining flour such that it will be challah once the dough is all formed. Making this stipulation circumvents the requirement that (according to the first reading) there be less than five quarter kav of flour remaining when he separates challah. That is because the separated portion only becomes challah for everything once it is all kneaded through.1
The Yerurshalmi explains that this was the practice taught to the kohanot. R' Chaim explains that people would bring the kohanot their flour, since the kohanot knew how to separate the challah in a way that would ensure the challah would remain tahor. The Mishnah Rishona notes that there is a risk that the separated portion might get burnt prior to the end of kneading which would mean that challah would need to be separated from the tameh dough undermining the decree. He answers that the only real concern was the dough becoming tameh, since it is being handled by many people. The separated portion however can be set aside carefully such that there is no real concern of it being comprised.
One might then ask, that if it only becomes challah at the end, it will not be considered as being separated min hamukaf (in close proximity to the dough) which is requirement when separating Challah. The Mishnah Rishona however cites that Rash that since it was separated and designated min hamukaf, even though it attains the kedusha of challah later, it is sufficient.
One might however also ask, how can one designate the portion as challah for flour that is not yet dough? It should be considered a "davar she'lo ba leolam". The Tosfot R' Akiva Eiger however answers that this case is different. Since one can continue kneading the rest of the dough, it is not considered as mechusar maaseh -- lacking a critical step. It is as if the requirement has been fullfill and "be'olam", and the declaration works.
The Derech Emunah (Bikurim 8:19) however notes that even though the Mishnah (see also Rambam 8:2) indicates that this is the preferred practice, that was because, as we have explained, they wanted to ensure the challah remains tahor. Nowadays, since all dough is tameh, the challah should be separated at the optimal time -- once kneading is completed.
1 R' Chaim (on Yerushalmi) explains that whether there still must be five quarter kav of flour mixed with water when the challah is separated is the subject of debate between R' Yossi and R' Yonah.
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