It's your first day of organic chemistry lab. You're doing liquid-liquid extraction. It's your first time that you have no clue what's going on, and the first time that you get a 216% yield. So, what went wrong? And how does that lab technique actually work? And more importantly, what you care about, how are they tested on the MCAT? okay i got an 896 percent yield and i submitted it if you're in this program cp6 high yield what are we covering There was so much high yield material in this. It was insane. It's chromatography and separation techniques, which is what we're covering today. But it also had like nucleic acids, amino acids, protein structure, and even intermolecular forces, which was more of a mid yield thing. But I am not going to go over all of those in the same video. He does not remember. I don't remember a single bit of it unless I prep it for these videos. I'm reading. I'm just kidding. Prompter. um but anyway today we're going to cover chromatography and separation because we've covered um all the other things in previous videos and if we haven't we're going to cover it in future videos sound good john oh yeah let's roll all right All right. So this basically talks about all the types of chromatography and separation. And what I wanted to do with this slide is basically just to show you how many types there are. That's why this topic is so confusing. Okay, you got liquid-liquid extraction, distillation, column chromatography, that includes ion exchange, affinity, size exclusion, gas chromatography, TLC, high pressure liquid chromatography. you do not have to be a master at all of these to get questions right on the MCAT. Okay. Yeah. And some, some like people, some practice exams, some third-party exam vendors, they will like test some of those really, really hyper-specifically. Like I know that Altius, they're my favorite third-party exams. but they test high pressure liquid chromatography. Like it is their dying mission. And it's, let me tell you, I have never heard of that. I have never heard of that before the MCAT. Yeah. Yeah. No, me either. I've never heard of it since the MCAT, but the double AMC and what I've seen on test day is like ion exchange, size exclusion. And I think I've seen TLC. I've seen TLC and I've seen, um, cause I, I remember it has like to do with retention factor. I've, I think I've done questions about that. I may have seen a gas chromatography. I may have, but that might've been cause Altius, uh, that's another one that they kind of, um, I feel like, yeah, they hit gas a lot. Maybe distillation on the WMC. Maybe I've seen a distillation. Maybe fractional. Yeah, maybe. Let me tell you what you're not going to see. You're not going to see affinity chromatography. You're not going to see probably vacuum distillation. Just those obscure ones. You're not going to see liquid-liquid extraction. And if you are, you're really actually being tested on solubility. You're not being tested on liquid-liquid extraction. Loki, when you said liquid-liquid extraction in the intro, I was like, ugh, that's a retake. She misspoke me. Like I didn't know it was a thing. I was stuttering yeah I was like this idiot how did you relate I love I loved it I love doing them because I like like inverting the little thing and then you got to worry about the glass blowing up and so I remember specifically I almost blew up y'all know what I'm talking about I almost blew up the the glass thing in a liquid liquid extraction because I didn't I forgot to like release the gas moving on moving on what's the point you're talking about now thank you okay yeah you got distillation but i know what you're talking about now yeah you separate like the organic layer from the um um inorganic layer inorganic i got you i got you shawty there we go okay you want to talk about you want to talk about yeah let me talk about chromatography and separation so Whenever we're talking about chromatography... It says molecules are often in solution with other molecules following chemical reactions. We can separate these molecules based on some physical characteristics that differ between the molecules in solution. So what this is saying is that in chromatography as a whole, we're just taking advantage of the fact that basically... molecules that have similar physical capabilities and similar physical capacities are going to stick together and so we can separate them based off of that yeah and i will say occasionally you'll see um a wrench thrown into that like how um acids dissolve in basic solutions and so you might see some of that play into it uh but typically like we're dealing with polarity or something like that or or boiling point or charge where same sticks with same you know yeah and i really on here go ahead I was just going to say, I almost put on here, like, I actually had it written out, like polarity would be like a thin layer of chromatography and boiling point would be gas chromatography, but it got real wordy. So we're only really going to point like to what you're separating or what you're basing your separation on for the specific chromatography techniques that we're going to talk about. So don't worry about that. you should though, you should have a flash card that knows, you know, like what, what does TLC use? You should know how that separates things because they're going to ask you to calculate an RF value, which is essentially how far your solvent is going to climb that piece of paper. And you need to know, oh, you know, based off of polarity, which one would run further. Yeah. We're just not going to dive too deep into it. Words. I agree and speak of the devil thin layer chromatography the first one up um and that's probably because it's the most simple and it's it's widely applicable and that high pressure liquid chromatography that we were talking about earlier the reason I I don't give a crap about it is because it has the exact same rules as thin layer chromatography and so I always just kind of like thought about TLC and that when I was presented with high pressure. It separates based on polarity. in the typical TLC setup, which I kind of have in a picture at the bottom, the mobile phase. Okay. When we talk about chromatography, we always have a mobile phase and a stationary phase. The mobile phase is typically a solution that moves and the stationary phase is typically, you know, in TLC, it's a silica sheet, like a sheet of silica that you literally sit in the mobile phase. The mobile phase is typically nonpolar. A lot of times it's something like hexane, some kind of solution like that. Whereas the stationary phase, like I said, is going to be that silica paper. If you're not familiar with it, basically you dot things, you dot different solutions. So probably these would all be known solutions and this will be like an unknown solution. and you dot them and you see how the different components that are in that unknown solution separate based on polarity. So you can see this is like a brown dot at the bottom, but once it separates out, you know, part of that brown dot, like it has obviously four different chemicals in it. Okay. And one of the chemicals is pretty polar so it doesn't move up with that hexane uh then as you go up they get more non-polar john i know i'm explaining that crappy help me out no you did a good job the the i remember doing this in lab at ole miss and what we did is we took like some you like some NSAIDs, some common painkiller, or yeah, some common like over-the-counter NSAIDs and stuff like that, anti-inflammatories. And then we had one vial that was a mix of all of them. And so let's just say purple was Tylenol and green was ibuprofen and red was aspirin. gray at the bottom right above three would have been a mixture of all of them. And so it would actually separate out into the three. So you could, it would stain specific dots at each of the three, and that would be the specific components of that mixture. So it's a really, really cheap. way. That's one reason it's so popular in the chemical field, but it's super easy to do thin layer chromatography. But I think you did it well. I mean, you've just got to, there's some basic chromatography principles like mobile phase moves, stationary phase doesn't move. You know, it's funny how that works. And the thing that's unique to TLC though, is retention factor. And so you do need to associate retention factor with TLC because that is not, to my knowledge, that's not associated with any other form of chromatography. I don't think so. There's retention time in gas chromatography, but that's not the same thing. And to, to, okay. So I got some terms here that are common terms when you're talking about the certain types of chromatography. So thin layer chromatography, like John just said, retention factor is a huge, uh, term that's thrown around also solvent front. And these two go hand in hand. What the solvent front is, is where the solution ends up. So you know, I've set my silica plate into my hexane. Originally it's only like up to this line right here, or maybe even a little bit below it. John, can you see that color? Yeah. Yeah. It being the mobile phase, the liquid, by the way. Yeah. so it would be like right here i'll make it red um and then through capillary action it would move up that paper that's kind of the whole point behind tlc so it only moves up to a certain point though and that point is right here in the picture and it's called the solvent front so to calculate your retention factor uh you take oh gosh i've gotten myself into some deep water trying to pull out of my memory equation for attention i'm so glad you didn't ask me to do it now you get the electric thing for fun um i'm pretty sure um it's the length of you know let's take this this um this purple dot right here um it's like the length from the center of the dot down to like the spotting line uh divided by the entire length of from the dotting line to the solvent front. That might be backwards. Y'all fact check me, but I'm almost positive that's it. I think that's right. I have a note here. Effect one greater than one. Yeah, that's exactly right. So I explained it right. Effect one greater than one. Effect two greater than one. I thought about capillary action this week and I went to the fair and because I hate my gallbladder and I had some cotton candy and I threw it at like a little puddle of I had I thought it was water then but now reflecting on where I was it probably wasn't water but um it was it was like carny juice or something heroin but yeah so like just a little bit of the cotton candy was touching the water and it just kind of sucked it all up yeah happily reaction i never forget it i got a quick story about a carny john one time i was at a carnival in ponticin mississippi where i'm from and uh that's you First off, don't get on any rides at a carnival in Mississippi. No, they're all attached. They are all attached to a pickup truck. Very visibly. Yeah, exactly. Like as you ride it. But there was a carny and he was not smoking a cigarette, but he opened his mouth to speak at me and smoke came out. He was not holding a cigarette. He had not. I've been watching him. He had not just come back from a smoke break. I was like, he's a dragon. or oh my gosh he's literally like expelling smoke like his blood is more smoke than it is co2 you just gotta start a slow clap at that point i know i was like oh my god anyway um I have a note here that thin layer chromatography is not used for separating entire solutions. We talked about liquid-liquid extraction. If you remember anything about that, that is used for separating entire solutions. This is not. This is for identifying how many molecules are in your solution by separating a very small sample, just like John was talking about, about those NSAIDs. I want to mention something real quick too here. because it's applicable to every type of chromatography, TLC can be reverse phase. That just means that in that case, the mobile phase will be polar and the stationary phase will be nonpolar. But what will the MCAT passage say in that case, Maggie? It'll say it'll be reverse phase. A lot of times it'll say the mobile phase is polar and the stationary phase is nonpolar. Yeah. At the very least, it will literally say reverse phase. So don't worry that like you're just kind of like, like they're not going to give you like some kind of obscure thing and expect you to know what the phases are. this is the yeah or like some go ahead yeah this is traditional oh my gosh and then if they say reverse phase just flip it and that's true for all these chromatographies All right, I give an honorable mention to gas chromatography just because we do like Alteas here and they do have it a lot on their practice exam. So if you want to pause and read this, because I went ahead and used my, you know, five minutes of my life to make this slide. So if you want to pause it and read it, that's fine, but we're not going to go over it. ionic exchange ionic exchange chromatography does not separate things based on polarity it separates things based on charged so what is the mobile phase it's charged liquid okay we're going to get into it and then the stationary phase is uh charged polymers so let's um polymers would be like just little beads by the way Yeah. Maggie and I, whenever we were prepping this session, we were talking and chromatography as a whole is just so like convoluted because there's so many like fancy science-y terms and stuff like that. But it's really easy when you boil it down. I mean, like TLC, you're just sticking a piece of paper in some liquid. And if it's similar in polarity to the liquid, it's going to rise really high. And if it's not, it's not going to rise that high. ion exchange, you got some beads that are charged, and if your liquid is the opposite charge of those beads, it's going to stick. You really do need to try to simplify this stuff to where you could teach it to someone that didn't quite understand the terms that you're using. Yeah, exactly. And that's why chromatography is so hard. Like even the word chromatography is too many syllables. But like if you kind of, you know, simplify it down into layman's terms I've drawn over here on the left hand side of the screen. kind of what it looks like. I mean, it's literally like a column, just like, just like this, but I just kind of redrew it up here so that we can see the charge of the polymers. Imagine they're literal beads in there. Okay. And you pour your solution through these beads. And just as John said, if it's negative, obviously it's going to stick to those positive beads. So what's going to run through, you know, if you have, let me zoom in on my picture here. this is my sample mixture it's got a bunch of different stuff in it okay negative positive neutral it's got a bunch of different stuff but we're trying to get negative stuff okay we're anion exchange if you could say so as it goes through these beads it's gonna separate itself based on charge so the the positive stuff is gonna be like oh don't touch me polymers and go straight through okay so this first elutant is what it's called and when it elutes out it comes out of the spigot or whatever um that's going to be your positive charge yeah that's a nice one i thought about it right just now and then you're going to have sort of a neutral stuff. It's going to be like, I don't really care about these beads. I'm bumping into them, but whatever, I'm not going to avoid them like the positive. I'm just going to kind of come out and I'm going to be neutral. Okay. That's like this purple right here, but the negative stuff is going to hold on to those beads. It's going to stick to them. So it's going to stay there for literally like a longer amount of time. Um, and eventually you're going to have to wash it out with a salt solution. to basically have something that likes that positive charge even more than your negative charge that you're selecting for. So probably like an even more negative molecule. More electronegative molecule. That's why they're just straight up electrolytes. And so they're going to bind that bead a lot tighter. Yeah, they're basically going to be like, get out here. This is my polymer. And so finally your negative solution is going to be washed out. And that's what you're going to be left with at the end. All right. I used a, I used a word here. I used a lutein. That's a term that's common in all the column chromatographies. Column chromatographies are chromatographies that happen in a column that looks like this. So that would be ion exchange, affinity, size exclusion. It's just kind of an overall catch-all term, but ion exchange specifically. What else? So the ion exchange chromatography can be broken down into two separate categories. And the categories just describe the charge that you are selecting for. Not the charge of the polymers, but the charge you are selecting for. cation exchange and anion exchange chromatography. And that's the way it'll be written on the MCAT. You'll see an anion column. They won't say an ion exchange column that uses negative beads. No, they will say a cation exchange column. And so... look out for those, you know, that, that's definitely something you need to make a note of. And I don't think it would be tested, but the, the way that you will like allude out progressively, more, I guess in our case, positive ions is that you're progressively, you're washing them in between each vial that gets taken with a salt solution. It's just getting progressively saltier. And so the concentration of salt will increase as you do continuous washes. Yeah. So that's why you see all these vials in the middle that are basically just like I mean, like literal, like salt water, um, or maybe like a salty non-polar solution that wouldn't affect anything else. But, um, yeah, just like John said, they get washed through every time to make sure that you're selecting for a single molecule and not just like some that kind of got stuck and don't actually belong in the fully negative charge, uh, solution. All right. Um, John, anything else? I don't think so. oh this one is used for separating entire solutions unlike tlc which was used for just identifying separate solutions in a dirty sample or whatever you want to call it this is used for separating entire solutions this is like my favorite chromatography it makes you can explain it then Okay. It separates molecules based on size. And this is used for, again, separating entire solutions. So you got a solution and it's got a bunch of different sized molecules in it. So you want to separate based on the size of you want to separate based on size your mobile phase is going to be the solution at large so you're just like dumping your solution into this size exclusion chromatograph and then your stationary phase are porous inert beads inert means it doesn't react with anything and porous means you know if this is the bead like it kind of blew up the bead here it's got these like canals in it okay and the canals are different sizes you can see this one over here on the right is pretty thick okay but this one right here is smaller it's thinner and that's what they look like in the size exclusion chromatography so you can imagine if you're pouring in this that has a bunch of different size molecules in it just like this picture shows i love this picture those those big molecules cannot get in those little crevices and so they completely like bounce around the porous beads and they actually elute first whereas the medium-sized beads they can go through some of these tunnels but they can't go through those smaller ones and so they kind of have to take some time to go through those porous beads they elute second those small or those yeah those small molecules you they take their dang time. They have to go through what's called like, I mean, they have to go through a very complicated route is typically the way that it's said. They have to go and the beads will probably be even more porous than this. They'll have stuff going everywhere, but those small molecules will have to go through all those little tunnels just because they are small enough to fit through it. So gravity is going to pull them through and they will elute last. there's no like reverse phase for this. This is the way that size inclusion chromatography always runs. Your big molecules elute first, your smallest molecules elute last. You do like it. Yeah. You explained. I do like it. I really like it. You look like you're sweating. It made no sense to me. That's because my AC's out, John. Let's get to some questions. Yeah, literally. Also, it's called gel filtration chromatography occasionally, but not that common. All right, John, me or you? Me. Go for it. Okay. It says researchers use reverse phase high performance liquid chromatography. You see what I'm saying? polar mobile phase and nonpolar stationary phase, okay, to separate compound two from compound one, which SEMA accurately describes this process. Okay, so a couple things. We got reverse phase, so it tells me that my mobile phase is polar and my stationary phase is nonpolar. to separate compound two from compound one. Those were given to me in the passage. Here we go, that's better. Those were given to me in the passage, so I'll put them right here. You didn't need to know anything else about the passage. You just need to know these two molecules right here. So what are some things I note? I know that high-performance liquid chromatography is a really great analog with TLC or thin layer chromatography. So I'm looking at polarity, okay? So I want to look at basically what's the difference in the polarity between these two before I go even into the answer choices, I noticed that compound to the only difference is that it has this alcohol group right here, that's the only group. And so that makes it more polar than compound one. Okay. So, I'm more polar in compound two. That means compound two is going to stick with the mobile phase more. I hope that makes sense to everyone because the mobile phase is the polar one. And compound one is going to stick more so, slightly more so, because it's just one alcohol group difference, right? But it's going to stick more so with the nonpolar stationary phase. Oops. Okay, so the answer choices say, A, increasing the polarity of the mobile phase will decrease the retention time of compound one relative to compound two. All right, well, if I'm increasing the polarity of the mobile phase, I'm not fully. I'm not fully convinced that I'm just going to decrease the retention time of compound one. And I say just compound one because it is relative to compound two. So I don't think it's going to like, I think it's going to have the same effect on compound one and compound two compound two, still going to stick a little more with the mobile and compound one still going to stick a little more with the stationary. So I don't know. I'm going to say maybe on that one. Whoops. B says compound one will elute first because it is more polar than compound two. No, it's not. Right. C, decreasing the affinity of compound one for the stationary phase will increase its retention time relative to compound two. No, because retention time would be how long it stays in the stationary phase. And so if you decrease the affinity of compound one for the stationary phase, it's not going to stick with the stationary phase for a longer amount of time. So I don't really like that one. D says compound two will elute first because it does not interact as favorably with the stationary phases compound one. Yeah, absolutely. That basically just means that compound two is more polar than compound one, which was our first thing that we realized. Yep. The hard part about that question is just keeping everything straight. read it very carefully as Maggie did me no no no no i was like i thought i read it pretty carefully no no no you you did well i was trying to give advice yeah yeah okay um me or her john i guess i'll try it i'm a little nervous the relationship between the steroid hormone estrogen and the peptide hormone insulin is being investigated In order to quantify levels of each of these hormones, tissue samples were homogenized and then placed in a mixture of 2 to 1 hexane to water. So hexane is more nonpolar. So I'll hit you with this MP. And then water is... Girl, I got you with the writing. Don't even try with that annotate. I feel like I'm pretty good with it. This is way better than my actual handwriting. Actually, it's... It is! What is the expected result from this extraction method? Okay. Let's see. So... I'm sorry, Siri is talking to me. Okay, so A says estrogen would be in the hexane phase. Insulin would be in the aqueous phase. Okay, I see what they're saying now. So estrogen is a steroid hormone, which means that it's going to be more nonpolar. In P, who would be? And insulin is a peptide hormone. And a characteristic of peptide hormones is that they are aqueous, meaning that they're going to be more polar in nature. Okay, so A says estrogen will be in the hexane phase, insulin will be in the aqueous phase. I don't hate that. I'll say maybe A. B says both estrogen and insulin would be in the aqueous phase. No, I don't like it. I do hate that. Because estrogen is a steroid hormone, which means it's made out of lipids, which means it's not aqueous. So say maybe not to be. C says insulin would be in the hexane phase. I dislike that already. And I feel like I am catching on to a trend here. D says, yeah, let's hurt its feelings. I like that. D says both estrogen and insulin would be in the hexane phase. That one's stupid too. How can we hurt its feelings? I got an idea. I got it. Let's make it so irrelevant that it's not even on the PowerPoint. It's not even an answer choice. So I don't like that either. So basically all this was saying is, hey, you need to pick up on the fact that steroid hormone means nonpolar, peptide hormone means polar, and just decide which of these would belong in which phase. So I would go with answer choice A. and a is correct you described that very well john thanks i was very nervous you know how you did you see did you guys see how john had to get a little bit of information from the answer choices to be able to simplify this question and really understand what it's asking that's okay like i feel like more than 50 of my questions i have to peek at the answers to even see what it's trying to ask me um but still some find the questions tim is always going to help All right, I'll take this. A protein with which properties will most likely have the largest negative net charge at pH 7? Okay, we got a protein and we're looking for a large negative net charge. um let's see a says a protein that binds to an anion exchange column at ph7 so anion exchange that means we're selecting for anions and we have positive charged beats A protein that binds to an anion exchange column at pH 7 and requires a high concentration of NaCl for elution. So that means it's going to be sticking to those beads pretty good. So I like that answer. A says a protein that binds to an anion exchange column at pH 7 and requires a moderate concentration of NaCl for elution. Okay, so basically the same thing as A except not as strong of a negative charge. So. I don't like that. I'm looking for the largest net negative charge. Approaching that binding spoke add-on. Strong of a negative charge. So if it only requires like a moderate concentration of NaCl for elution relative to a high concentration, that means that it's not going to take that much negative charge to push it out because it's not a very negatively charged protein to start with. I love it. Okay. Um, a protein that binds to a cation exchange and cation exchange selects for cation. So that would be selecting for a positive charge. Girl, what does that say? Oh, stupid. I thought you were writing selects for cations. I was like, it ain't even close. Um, D a protein that binds to a cation. Okay. Okay. I didn't know that I could do this on your screen share. We're just learning. Really? Okay. Well, these sessions are never going to be the same now that you found that out. No, no, no, no. They will not be deductive either. And no one will ever be able to read anything. All right. That's the end of the question, guys. Thank you so much for... logging on with us and talking about chromatography, which is kind of a really boring topic in chemistry, but we hope that we made it fun and easy and manageable. We can't help you with the percent yield though. Oh, no, no, no, no, no, no, no, no, no, no, no, no, no, no, no, no, no, no,