Mixtures of amino acids can be analyzed by first separating th...

Question

Mixtures of amino acids can be analyzed by first separating the mixture into its components through ion exchange chromatography. Certain amino acids placed on a cation-exchange resin containing sulfonate groups (—SO₃^-) flow down the column slowly because of two factors that influence their movement: (1) ionic attraction between the sulfonate residues on the column and positively charged functional groups on the amino acids, and (2) hydrophobic interactions between amino acid R-groups and the strongly hydrophobic backbone of the polystyrene resin. For each pair of amino acids listed below, circle the amino acid that is eluted first from the cation-exchange column by a buffer at pH 7.

Accepted Answer

Alright. So at first glance, this practice problem looks really, really challenging because it gives us so much information. But really, all it's asking us to do is for each pair of amino acids that's listed down below, it wants us to circle the amino acid that elutes first from the chromatography column. And it tells us that we're specifically using cation exchange chromatography columns. And so all we need to do is look at each of these pairs of amino acids and circle the amino acid that elutes first or comes out of the column first. So that's pretty easy to do. And so, recall that with cation exchange chromatography, which I've drawn over here, that it uses negatively charged stationary resin, which are these, highlighted, negatively charged blue balls that I'm showing here. And we know that positively charged proteins and amino acids, which are in red, are gonna interact the strongest with the negatively charged stationary resin. So we can see them interacting really strongly, and they're not going to move as much as all of the other amino acids. So because they are going to not move as much, they're gonna stay in the column longer and they're going to elute last from the column. So with the positively charged amino acids, recall from our previous lessons that these are gonna be the ones to elute last. So as a reminder, we'll put elute last over here for the positively charged amino acids. Now, the negatively charged amino acids on the other hand are essentially going to repel that stationary phase. And because they repel the stationary phase, they're not gonna interact with it at all and they're going to flow right out as fast as possible with the mobile phase. So these negatively charged proteins and amino acids are going to be the ones to elute, first from the column, so they will elute first. And so as a reminder from our previous lessons, we'll put elute first. And then we know that the neutral amino acids which are in between, that they are going to essentially elute in between the 2. And so, what we can do is we can put that these will elute second. And so, you can see here that we've got the positively charged proteins at the top of the column, the neutral proteins and amino acids, in the middle, and then the negatively charged amino acids and proteins at the bottom because they elute first. And so, really, that's all the information that we need to recall from our previous lessons to answer this problem. And all we need to do is compare each amino acid and figure out which one of these groups does it fall into and which, which one's going to elute, first. And so looking at our first pair here, we have aspartic acid and lysine that we're comparing. And aspartic acid's one letter code is d and lysine's one letter code is k. And from our mnemonic, dragons eat knights riding horses, we know that lysine or, I'm sorry, aspartic acid is a negatively charged amino acid, and lysine is a positively charged amino acid. And so, because the positively charged amino acid of lysine elutes last from the column, and the negatively charged amino acids of aspartic acid elutes first from the column, that tells us that we can circle aspartic acid since it's going to elute first here. And so all we need to do for, part a here is circle aspartic acid. And that tells us that, that is going to be the one that elutes first in this pair, and that's it for part a. So pretty easy. Right? All we did was apply those same rules that we already knew from our previous lessons. And so we're gonna do the same for the rest of these parts here. And next, what we have is, part b, where we're comparing arginine and methionine. And arginine's one letter code is r, and methionine's one letter code is an m. And what you'll see is that methionine is a nonpolar hydrophobic amino acid with a neutral charge. And neutral amino acids are going to elute second, whereas, arginine is a positively charged amino acid, and positively charged amino acids elute last. So that means that methionine is going to elute first, and we can go ahead and circle methionine as being the correct answer, since arginine is positive and it loops last. So that's it for part b. And now we can do the same for part c. So pretty easy, right, so far, and we're just going to keep moving through. So glutamic acid and valine are the 2 amino acids that comparing. Glutamic acid's one letter code is an e, and valine's one letter code is a v. And what you'll see is that valine is, again, a nonpolar hydrophobic amino acid. And it's neutral, so it's going to elute second, whereas glutamic acid is, a negatively charged amino acid, and so, because it's negatively charged, it's going to elute first from the column. And so all we need to do is circle glutamic acid, for this problem, for this part, because it's going to elute first from the column since it's negatively charged. And so now we're moving on to part d, and in part d, we're comparing glycine and leucine, and glycine's one letter code is g, and leucine's one letter code is l. And what we'll see here is that both glycine and leucine, fall into the non polar hydrophobic amino acids, and they are both neutral amino acids. So now all we need to do is use another level of, comparison between glycine and leucine. Since they fall into the same group, we need to figure out, what is it about their r groups that allows one to elute first before the other? And so, if you remember, glycine's r group is literally just a hydrogen. It glides right on through. And leucine's r group is a loose extension of valine, so it's CH 2, and then you have the valine at the end with 2 methyl groups. And so, essentially, what you'll notice here is that hydrogen is literally such a small r group, but leucine here has a much larger r group with more hydrocarbons. And more hydrocarbons means that it's going to be even more non polar and more hydrophobic than glycine. And notice up above in our problem, it tells us that hydrophobic interactions between the amino acid r groups and the hydrophobic, backbone of the resin, the stationary resin in the column, is going to be one of the two factors that will slow the amino acids down. And so because leucine has a more hydrophobic amino acid r group, I'm sorry, it has a more hydrophobic r group, it's gonna be able to form stronger hydrophobic interactions. And stronger hydrophobic interactions is gonna cause it to move more slowly through the column. And so essentially what that means is that leucine is going to elute last here. And because it's going to elute last, that means that glycine is going to elute first. And so we can go ahead and circle glycine here as the correct answer. And so that is it for part d. Now moving on to part e, we have serine and alanine that we're comparing. And serine's one letter code is an s, and alanine's one letter code is an a. And what you'll notice is that serine is a polar amino acid, and alanine is a non polar hydrophobic amino acid, but they are both categorized as neutral amino acids, since they don't fall into this charged amino acid group. And so we need to look at their r groups to distinguish which one of these 2 is going to elute first from the column. And so, recall that the r group of serine is literally just alanine, so it's alanine with an alcohol group. So you remember alcohol is a serious threat, so we know it has an alcohol group, and so it's gonna be, serine, CH 2 Oh, that's serine to R group. And alanine's R group is literally just going to be a methyl group, it's just a CH 3. And so what you'll see here is that, serine has this polar group at the bottom, this hydroxyl group, and we know that the oxygen is essentially gonna have a partial negative charge on it. And so, alanine, on the other hand, is not going to have any partial charges. It's not polar. It's all nonpolar interactions. And so because serine here has this partial negative charge, it's acting more so like a negatively charged amino acid. And, for that reason, it's going to actually elute before alanine. And so, what that means is that serine is going to elute first. And that's because, again, it has the partial negative charge on its hydroxyl group, and, that's gonna make it act more like a negatively charged amino acid and elute first. And so that concludes this practice problem, and although it may have seemed, very very challenging at the beginning, we broke it down into these very basic components from our previous lessons, and we were able to determine the correct answers. So that concludes this practice problem, and I'll see you guys in our next video.

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