Chromatographic Separation #2
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Nielsen, Kristian
N. N.
Chromatographic separation; Chapters:
 I don't know if you all managed to get through the exercise. Deliberately, it is difficult. Did some of you manage to get through it? Yeah? So the purpose of this exercise is actually to put in the compounds on the scale that we had before. So again, here we got the scale from polar to apolar. And the point is that it's fairly easy, as soon as you know the principles, to put in the compounds in the extremes. So recognize the really polar compounds and to recognize the really apolar compounds. And all the things in the middle, they can actually be quite difficult to predict. And I talk to some of you and you say, oh, you can't see the difference. How on earth do you see the difference? You don't. And this is exactly why we got more than one separation principle, because sometimes it's not easy to predict. So if we can exploit a charge or a size instead, that might be more optimal. But the reverse phase is usually DC18 change. So they are sticking out from the particles and the interaction from your molecules to this is very, very fast. And they're moving out here and the reverse phase is the fast principle here. This is why it's usually the most efficient. So this is where we can really see minute differences for molecules. Yeah, but we're not able to predict them. Not very well, not always. So did any of you have an idea when we were looking at pH2, which of these compounds represent the most apolar one? Which is the further right end of the scale. Any ideas? I know some of you got de-answered. What is your? Say A. Yeah. And why is that? Because it has this big A polar arc. Yeah, so it's got this long aliphatic chain which fits like a hand in a glove with the C18 that Christians show before. So even though it actually got this sugar unit sitting on here, it doesn't matter compared to all these carbons sitting out here. And yeah. This is an amide, so no charge. Yeah, exactly. So this compound won't be affected by charge either at low or high pH because amides are not particularly good at accepting extra protons. So and also this hydroxyl groups, it's sitting in beta position to the double bond. So that will also hold on quite tightly to its proton. So it won't be affected at all. So this will actually represent the A polar end of the scale both at high and low pH. If we try to look at the other end of the scale, if we take the low pH, which compound will be the most polar one? Most soluble in water. Ideas? Yeah, I heard that you got to give it a first. Yeah, a few monosin. And you also had the explanation you told me. Yeah, because of the four acid groups. Yeah. So it got these four acid groups sitting out here and they will contribute to the polarity of the compound. It's actually here. Yeah. So you got this beautiful compound here. They sit around. Yeah. So they will be charged at high pH. And here out in the end we got a primary amine, which will be charged at low pH. So this will have a charge both of pH 2 and pH 11 and this will be polar at all times. So this is the other end of the scale. But four negative charges. Yeah. So if you go high, means a lot more than one. Yeah. And the point is that even though you got that aliphatic part here in the middle, when you actually see the molecule, you can see that the TCA groups, the tricarboxylic acid chains sitting out here, they can fold in and cover that part of the molecule. So this was when I talked about 2D structures and 3D structures. This compound will fold up and be even more polar. So when we got the 3D structure. This is the other end of the scale. And this is now we got the fourth remaining structure. This is where it gets difficult. What do you think that happens if we then remove a hydroxy group here? They come out before or after when it intact with this. Yeah. So yeah. So when you remove, that's where you got the little table there. So you exchange an OH with an H. Then you'll see a positive contribution to your log C value, which means that it will probably elute later. It will be more apolar. That could be some of the things we will discuss to the exam. If we have a lot of isomers you worked on here, let's say one of the teams get this as the biosynthesis. Then you should know that if you add hydroxy group that will come early on reverse phase. If you remove some, they will come later. That's the kind of thing we will ask. We won't throw you six structures and say now we want the order. If we do, then it will then new up at the very high end of the grades. Yeah. If you can answer this one, you'll probably pass right away. Yeah. So we got the four here in the middle. They're all what we'll call medium polar. So here we got a Pender Brumusudolin. I got five bromines. Bromines will contribute to a positive log D value. It will make the compound a lot more apolar. So this is one of the next ones coming after this one up here. But in fact, at high pH, it got a phenyl group down here, which means that it can be negatively charged. It's a weak asset. So this will actually change polarity at high pH. It will become more polar. Then we got a compound here. It does not have any groups that can carry a charge. It won't be affected by changes in pH. So it will lie in the middle more or less no matter what you do. Where exactly in the middle is not the point of this exercise. But it will be unaffected. Then we got this funny compound up here. Christian will have a case for that later on. Because sometimes polarity and solubility and retention is not always the same. And this goes for this compound here. But if you go look only at the polarity, the log D value, this phenyl up here charged at high pH. And then it does some funny stuff. So even though it's an amide here, the double bond here, it can move around electrons and actually accept a proton. So this will be positively charged at low pH. So even though it's actually a quite large molecule with a lot of aromatic systems, it's actually quite polar at low pH. And the last molecule here, you got an enol, which has the same effect as a phenyl. It's a weak asset. So the electrons here of the double bond, they will pull away the electrons of the bond between the O and the H. High pH. We got a negative charge. And actually the pKa of this one, this extreme example, this is a true example, it's the same as a carboxylic acid. So it's about 4.5 here. Normal chemists wouldn't believe that. But that is actually the case. So a little bit of a difficult exercise, but hopefully you'll get a feeling of movements and relative polarity. That would be much more. Yeah. So when we were talking about separations based on polarity, we got free separation principles, so for types of chromatography you're going to learn about. We got the reverse phase, normal phase, and helic, hydrophilic interaction chromatography. And they're good for different types of compounds. So we got the scale before. Reverse phase is a very versatile method. It's got a very high polarity range. So it can actually hold on to quite polar compounds and it's able to separate very apolar compounds as well. So this is a really good starting point. The order of how molecules move through the column, moving through the street of bars. You'll got the polar ones, they will come out first because the stationary phase that Christians showed here as well. C18, it's very apolar. That will attract likes, so it will attract apolar compounds while the polar ones will be more quickly pushed through. So we'll have the polar compounds coming out first and then apolar coming out last. The stationary phase, what's sticking on the beats or the particles? C18, as we talked about already, but you can also have other types of functional group. If the molecule sticks too well, we could, then of course get a C8 or a C4. It's very easy for the chemist to make these. So that changed the polarity of the stationary phase and that of course changed the selectivity towards the polarity of the compounds coming through. The solvents that we use, the mobile phase that we use to pull compounds away from the stationary phase is water, acetonitrile and methanol. These are the compounds, the solvents on the left side on the scale, the polar solvents, most of course water, and then acetonitrile and methanol. And these solvents are really good, especially when we do detection with mass spectrometry because these solvents do not affect the ionization of these compounds. We'll come back to that later. So when we have a normal phase, as a reverse phase and normal phase, reverse phase is what we normally use and normal phase is what we not normally use. So it's very confusing, but this is the original way of making chromatography, that's why it's called normal phase. And this is actually where you got your separation of your A-polar compounds because your stationary phase is more polar. It's not decorated with all these C18 chains, we got the pure silica, you got the beat when you got the R-group sticking out, no R-group, just silica. So it's really good for fats and sterols and trigrocerides and all these A-polar compounds. And the order of elution is the opposite. So it's the most A-polar compounds, they will be pushed quickly for the column while the polar ones will stick longer because the stationary phase is more polar. So again, we use silica, but we can also put just some hydroxyl groups on the silica called a diol or amino groups. So all these polar groups, we can stick on the silica and attract likewise polar molecules. The solvents that we use for this are a little bit more harsh. So we use heptane, dichloromethane, ethylacetate and methanol. So these are the A-polar solvents. And this is one of the reasons why normal phase is not necessarily good for interfacing with MS, because, yeah. We need some water, actually. Water is good for making ions. So this is not something that you will usually connect with your MS or something like that. Another thing is that a lot of these solvents actually have great UV absorption, which again, like before the break, which means that we can't see the compounds that we wanted to take by UV. So it gives a lot more background. But it can still be very good for separating compounds, and then we have to use other types of detectors. And Christian will talk about that later this afternoon. Then we got hydrophilic interaction chromatography. And this is where we have the extremely polar to medium polar compounds. It's a more new method. If you've got normal phase, the old-fashioned way, this is the modern way, and this is the really modern way. So not a lot of people use helix, and not a lot of people understand exactly how helix works. It's one part chromatographic signs, and one part black magic. So, yeah. But still, it's good to know that you got the possibility. You'll be the ones moving chromatography into a new era where we actually know how to use this method properly. Like normal phase, it's got great affinity for the polar compounds. So the A polar ones will be pushed through the column much faster, and then the polar ones will stick. And the stationary phases actually resemble a lot the ones that we use in normal phase. So it's silica, amino phases, and we have switter ions, so different phases that can carry both positive and a negative charge at the same time. But the huge difference compared to normal phase is the solvents that we use. So in normal phase, we got the very A polar solvents, heptane, and all that we can't use for making ions. Oh, no, this one's up. While in helix, we can use water. Water is actually the basis of hydrophilic interaction chromatography, which means that this is a type of aqueous normal phase that we can use when we want to detect by MS. So we use water, we use acetonitrile, and we use different kinds of buffers, salt solutions to as a mobile phase for this one. No, it's just a laser not working on. Yeah, nothing. So you can actually modify your reverse phase stationary phase to interact with more polar compounds. So again, because helix is black magic, sometimes it's worth just looking at some of the more polar reverse phases. So that can be a phenyl, cyanoh, or pentafluorofenyl, or graphite. So sticking on the beat, you can have all these different types of functional groups, and moving from left to right, they are more and more polar. But graphite is also Moodoo, so that can do a lot of strange things. So that's... But it exists. It's always nice to know that it exists. Yeah. So depending on what type of functionality that you have on your stationary phase, you change your cell activity. And phenyl and pentafluorofenyl, they're not just more polar, they also have other types of cell activities. Because of the benzene ring. So the benzene ring here, that can create these van der Waals forces that we talked about last time. You had a molecule morphine, I think it was. So you got kind of a dipole here that will attract other aromatic molecules. So here you got the stationary phase with an analyte aromatic ring sitting on top. So that will hold on to aromatic compounds even harder. And also you have that dipole interaction in your pentafluorofenyl group, which also gives you a different selectivity. So if you got aromatic compounds with similar polarity to a non-aromatic, you can actually change that selectivity by choosing a different type of reverse phase column. Questions? When we're talking about the stationary phases in normal phase, again we have the amino, the diol, silica, and different amide functionalities sticking on that. And it's very, very important that if you want to use one of these stationary phases for normal phase separation, you don't put water anywhere near that. Because once you do that, you ruin that effect. You only use these very apolar solvents. Because when you got the silica, all these different cross links that you saw in between, they can be broken by water, they can be hydrolyzed, and that will change the chemistry of the particle. So actually when you work here, we also have some small one-time use columns we can use to purify. And in those, you have to dry them in an oven. And you will also see working in the summer, which is very humid, but they will not work as well as now where everything is dry. So also some that makes these, they actually add a little bit of water, so they know that it's 0.05% water, something like that. Simply to say, we cannot control water, so we add a controlled amount. But in general, we try to keep these as water-free as possible. Yeah. So. This also why this chromatography is not as popular as the reverse phase, because we have a reproducibility problem, because of moisture and all kinds of things. Yeah. So the difference, like I said, with normal phase and aqueous normal phase, HILIC, is that when you use silica or amino as a HILIC phase, you actually put water in there deliberately, because here you see that silica surface here. And actually when you put water in, all that layer will be filled with water. And that water layer is actually your stationary phase, or you can say, can you say that? Yeah. Yeah. So it's the water layer that is in here that is important, and not necessarily the functional groups itself. So when you have used hydrophilic interaction chromatography, you got an amino phase, and you got all that water bound in between the amino groups, sticking out from the silica, which means that when your mobile phase has a high organic content, so when you start out with acetonitrile, for example, the polar compounds, they are not fond of acetonitrile, they'll go in and hide here in the water layer. So they'll stick to that water layer with the stationary phase, and the mobile phase with high organic will just pass through and let them rest there for a while. So when you want in HILIC, you want your compounds to leave the bar, you want them to leave the column, you increase the water content, because then the compounds can diffuse out from the water layer into the mobile phase and leave the column. So here, this water layer is really essential for how that works. So we use water, we use acetonitrile, we don't use alcohols, because when you put alcohols in here, that will confuse everything and make a very weird layer in between, and we have no HILIC effect at all. So the water is the key part of this separation. So now we're going to start up whole, hope that works. So here you got a TLC plate, so it's called thin layer chromatography. Have you heard about that before? Yeah, very quiet. So here you got, like you got a column, you got a flow of mobile phase passing through, so the compounds move in this direction. And then we got these acylated homocerin lactones, these are bacterial signal molecules, bacteria that make them to communicate with each other and send war signals and signals to colonize the surface, and there are a lot of different analogs of these. And they all have this homocerin lactone head, and then they got an aliphatic chain here of different length. So in this case you got a very long aliphatic chain, so you got a compound that is very apolar or polar. Apolar, yeah, correct. So this is very apolar. This here on the other hand got a hydroxyl group, so it's polar. So apolar to polar, eluding with 60% methanol in water. What type of chromatography do we have here? So go vote and see how it works out. Click the bar on top. Yeah, I couldn't see it. I think it's good to have. Yeah, I think it's good to have. Hello. Yeah. Yeah. Stopping the poll. Anyone dares to tell me what they voted before I put on the distribution? Yeah. See if it works. That's beautiful. Almost 70% on reverse face. A few on normal face and HILIC, but the correct answer is in fact reverse face. So you got the most apolar compounds coming out first, and then you moved to the polar ones. Both normal face and HILIC, they'll have the polar compounds coming out first. And also if you're in doubt between normal face and HILIC, you can see that on the solvents because we added water, which is a no-no in normal face. So this is the way that you can recognize these different kinds of chromatography. This is also good for the ones who know what happened here is that we added the compounds, and actually then you draw the TLC plate, and if you don't draw it, then it doesn't work, because then it will kill the bacteria after that. Anyway, then you have the TLC plate, and we then have a argon at 50 degrees. When it's almost starting to solidify, and then you have a signal bacteria that can produce this blue color. Then you put it in, you shake it very fast, and you put it on the TLC plate, and you wait for about four seconds. Oh, yeah, three, four days, and then here you can simply see then it gets colored where you have the signaling molecule. This is also an example of where we interface the chromatography with biology and use biology as a detector. Yep. Yep. We'll move directly to a new poll. Now I'll give you some hints on how you can recognize the different types, and I'm just going to start that. So here again, we want to know what type of chromatography it is, but we will also, as a secondary question, you can vote two times. You can ignore the second part of the question if you want to, but which of these diacetyl glucose and glucose is peak number one and peak number two? So from the text up there, see if you can try to recognize the type of chromatography and then figure out whether or not the A-polar or the polar analog of glucose will come out first. And if you don't see the question on your computer, try refreshing the browser. I don't know how quickly it updates. And again, you can just ignore the second part of the question if you... Okay. Okay. Okay. Any final votes? Last one coming in. So, excellent. So you all guessed more or less that this was HILIC. You definitely... none of you voted for reverse waste, which is great. You realize that you started with a high organic content, so which means that it's either normal phase or HILIC, but you got water, which means that it's HILIC. And the order of these two, that is more difficult. So that's 50-50 on the votes. And this is because we both have polarity of the compounds and polarity of the stationary phase, and now everything is confused. But HILIC, we got the A-polar compounds coming out first. And which of these two are the most A-polar one? Come on. So, in this case, you got extra C-groups. You got acetyl sitting instead of hydroxy, which makes it more A-polar. So, this one will be the one coming out first, and this one will be the one coming out last. And this would be the opposite if we had reverse space. So, now I understand why you're confused. So, like we talked about, that we can predict these log D values. And log D predictions, they're good for prediction of retention time for some compounds. So, you got these homo-saryon lactones that you got on the TLC plate before. It's a beautiful linear relationship between retention time and log D value. So, you see, the longer the chain gets, the higher the log D value, the more A-polar of the compound, the longer the compound will stick to a reverse phase column. And recall here, this is actually calculated. This is not measured in a lab. This is just a calculated one. But also here, even though, let's say, it actually doesn't calculate it right or a little bit wrong, but then, you know, adding here still helps a lot. So, you know, it would only move it up. So, that's why it makes such a good correlation. So, these calculation tools are very, usually very good in one to predict a small modification. Yeah. So, actually, when you got a chromatogram like this, again, you can predict the order of retention based on polarity. So, now you got a lot of exercises in the printout. And I'm just going to go because I realize that the last exercise, this is the last exercise for this section. So, there's coffee on the table. There's cake on the table. You can move to the next room. And we'll give you what, half an hour, Christian? Yeah. Yeah. Half an hour to talk about the exercises and then we'll... Yeah. Coffee and sugar and everything should be next door.
 I don't know if you all managed to get through the exercise.  Deliberately, it is difficult.  Did some of you manage to get through it?  Yeah?  So the purpose of this exercise is actually to put in the compounds on the scale that we had before.  So again, here we got the scale from polar to apolar.  And the point is that it's fairly easy, as soon as you know the principles, to put in the compounds in the extremes.  So recognize the really polar compounds and to recognize the really apolar compounds.  And all the things in the middle, they can actually be quite difficult to predict.  And I talk to some of you and you say, oh, you can't see the difference. How on earth do you see the difference? You don't.  And this is exactly why we got more than one separation principle, because sometimes it's not easy to predict.  So if we can exploit a charge or a size instead, that might be more optimal.  But the reverse phase is usually DC18 change.  So they are sticking out from the particles and the interaction from your molecules to this is very, very fast.  And they're moving out here and the reverse phase is the fast principle here. This is why it's usually the most efficient.  So this is where we can really see minute differences for molecules.  Yeah, but we're not able to predict them.  Not very well, not always.  So did any of you have an idea when we were looking at pH2, which of these compounds represent the most apolar one?  Which is the further right end of the scale.  Any ideas? I know some of you got de-answered.  What is your?  Say A.  Yeah.  And why is that?  Because it has this big A polar arc.  Yeah, so it's got this long aliphatic chain which fits like a hand in a glove with the C18 that Christians show before.  So even though it actually got this sugar unit sitting on here, it doesn't matter compared to all these carbons sitting out here.  And yeah.  This is an amide, so no charge.  Yeah, exactly.  So this compound won't be affected by charge either at low or high pH because amides are not particularly good at accepting extra protons.  So and also this hydroxyl groups, it's sitting in beta position to the double bond.  So that will also hold on quite tightly to its proton.  So it won't be affected at all.  So this will actually represent the A polar end of the scale both at high and low pH.  If we try to look at the other end of the scale, if we take the low pH, which compound will be the most polar one?  Most soluble in water.  Ideas?  Yeah, I heard that you got to give it a first.  Yeah, a few monosin.  And you also had the explanation you told me.  Yeah, because of the four acid groups.  Yeah.  So it got these four acid groups sitting out here and they will contribute to the polarity of the compound.  It's actually here.  Yeah.  So you got this beautiful compound here.  They sit around.  Yeah.  So they will be charged at high pH.  And here out in the end we got a primary amine, which will be charged at low pH.  So this will have a charge both of pH 2 and pH 11 and this will be polar at all times.  So this is the other end of the scale.  But four negative charges.  Yeah.  So if you go high, means a lot more than one.  Yeah.  And the point is that even though you got that aliphatic part here in the middle, when you actually see the molecule, you can see that the TCA groups, the tricarboxylic acid chains sitting out here, they can fold in and cover that part of the molecule.  So this was when I talked about 2D structures and 3D structures.  This compound will fold up and be even more polar.  So when we got the 3D structure.  This is the other end of the scale.  And this is now we got the fourth remaining structure.  This is where it gets difficult.  What do you think that happens if we then remove a hydroxy group here?  They come out before or after when it intact with this.  Yeah.  So yeah.  So when you remove, that's where you got the little table there.  So you exchange an OH with an H.  Then you'll see a positive contribution to your log C value, which means that it will probably elute later.  It will be more apolar.  That could be some of the things we will discuss to the exam.  If we have a lot of isomers you worked on here, let's say one of the teams get this as the biosynthesis.  Then you should know that if you add hydroxy group that will come early on reverse phase.  If you remove some, they will come later.  That's the kind of thing we will ask.  We won't throw you six structures and say now we want the order.  If we do, then it will then new up at the very high end of the grades.  Yeah.  If you can answer this one, you'll probably pass right away.  Yeah.  So we got the four here in the middle.  They're all what we'll call medium polar.  So here we got a Pender Brumusudolin.  I got five bromines.  Bromines will contribute to a positive log D value.  It will make the compound a lot more apolar.  So this is one of the next ones coming after this one up here.  But in fact, at high pH, it got a phenyl group down here,  which means that it can be negatively charged.  It's a weak asset.  So this will actually change polarity at high pH.  It will become more polar.  Then we got a compound here.  It does not have any groups that can carry a charge.  It won't be affected by changes in pH.  So it will lie in the middle more or less no matter what you do.  Where exactly in the middle is not the point of this exercise.  But it will be unaffected.  Then we got this funny compound up here.  Christian will have a case for that later on.  Because sometimes polarity and solubility and retention is not always the same.  And this goes for this compound here.  But if you go look only at the polarity, the log D value,  this phenyl up here charged at high pH.  And then it does some funny stuff.  So even though it's an amide here, the double bond here,  it can move around electrons and actually accept a proton.  So this will be positively charged at low pH.  So even though it's actually a quite large molecule with a lot of aromatic systems,  it's actually quite polar at low pH.  And the last molecule here, you got an enol, which has the same effect as a phenyl.  It's a weak asset.  So the electrons here of the double bond,  they will pull away the electrons of the bond between the O and the H.  High pH.  We got a negative charge.  And actually the pKa of this one, this extreme example,  this is a true example, it's the same as a carboxylic acid.  So it's about 4.5 here.  Normal chemists wouldn't believe that.  But that is actually the case.  So a little bit of a difficult exercise,  but hopefully you'll get a feeling of movements and relative polarity.  That would be much more.  Yeah.  So when we were talking about separations based on polarity,  we got free separation principles,  so for types of chromatography you're going to learn about.  We got the reverse phase, normal phase, and helic,  hydrophilic interaction chromatography.  And they're good for different types of compounds.  So we got the scale before.  Reverse phase is a very versatile method.  It's got a very high polarity range.  So it can actually hold on to quite polar compounds  and it's able to separate very apolar compounds as well.  So this is a really good starting point.  The order of how molecules move through the column,  moving through the street of bars.  You'll got the polar ones, they will come out first  because the stationary phase that Christians showed here as well.  C18, it's very apolar.  That will attract likes, so it will attract apolar compounds  while the polar ones will be more quickly pushed through.  So we'll have the polar compounds coming out first  and then apolar coming out last.  The stationary phase, what's sticking on the beats or the particles?  C18, as we talked about already,  but you can also have other types of functional group.  If the molecule sticks too well, we could,  then of course get a C8 or a C4.  It's very easy for the chemist to make these.  So that changed the polarity of the stationary phase  and that of course changed the selectivity  towards the polarity of the compounds coming through.  The solvents that we use, the mobile phase  that we use to pull compounds away from the stationary phase  is water, acetonitrile and methanol.  These are the compounds, the solvents on the left side  on the scale, the polar solvents, most of course water,  and then acetonitrile and methanol.  And these solvents are really good,  especially when we do detection with mass spectrometry  because these solvents do not affect the ionization  of these compounds.  We'll come back to that later.  So when we have a normal phase,  as a reverse phase and normal phase,  reverse phase is what we normally use  and normal phase is what we not normally use.  So it's very confusing, but this is the original way  of making chromatography, that's why it's called normal phase.  And this is actually where you got your separation  of your A-polar compounds  because your stationary phase is more polar.  It's not decorated with all these C18 chains,  we got the pure silica, you got the beat  when you got the R-group sticking out,  no R-group, just silica.  So it's really good for fats and sterols  and trigrocerides and all these A-polar compounds.  And the order of elution is the opposite.  So it's the most A-polar compounds,  they will be pushed quickly for the column  while the polar ones will stick longer  because the stationary phase is more polar.  So again, we use silica,  but we can also put just some hydroxyl groups on the silica  called a diol or amino groups.  So all these polar groups,  we can stick on the silica and attract  likewise polar molecules.  The solvents that we use for this are a little bit more harsh.  So we use heptane, dichloromethane,  ethylacetate and methanol.  So these are the A-polar solvents.  And this is one of the reasons why normal phase  is not necessarily good for interfacing with MS,  because, yeah.  We need some water, actually.  Water is good for making ions.  So this is not something that you will usually connect  with your MS or something like that.  Another thing is that a lot of these solvents  actually have great UV absorption,  which again, like before the break,  which means that we can't see the compounds  that we wanted to take by UV.  So it gives a lot more background.  But it can still be very good for separating compounds,  and then we have to use other types of detectors.  And Christian will talk about that later this afternoon.  Then we got hydrophilic interaction chromatography.  And this is where we have the extremely polar  to medium polar compounds.  It's a more new method.  If you've got normal phase, the old-fashioned way,  this is the modern way,  and this is the really modern way.  So not a lot of people use helix,  and not a lot of people understand exactly how helix works.  It's one part chromatographic signs,  and one part black magic.  So, yeah.  But still, it's good to know that you got the possibility.  You'll be the ones moving chromatography into a new era  where we actually know how to use this method properly.  Like normal phase, it's got great affinity for the polar compounds.  So the A polar ones will be pushed through the column much faster,  and then the polar ones will stick.  And the stationary phases actually resemble a lot  the ones that we use in normal phase.  So it's silica, amino phases,  and we have switter ions, so different phases  that can carry both positive and a negative charge at the same time.  But the huge difference compared to normal phase  is the solvents that we use.  So in normal phase, we got the very A polar solvents,  heptane, and all that we can't use for making ions.  Oh, no, this one's up.  While in helix, we can use water.  Water is actually the basis of hydrophilic interaction chromatography,  which means that this is a type of aqueous normal phase  that we can use when we want to detect by MS.  So we use water, we use acetonitrile,  and we use different kinds of buffers, salt solutions  to as a mobile phase for this one.  No, it's just a laser not working on.  Yeah, nothing.  So you can actually modify your reverse phase stationary phase  to interact with more polar compounds.  So again, because helix is black magic,  sometimes it's worth just looking at some of the more polar reverse phases.  So that can be a phenyl, cyanoh, or pentafluorofenyl, or graphite.  So sticking on the beat, you can have all these different types of functional groups,  and moving from left to right, they are more and more polar.  But graphite is also Moodoo, so that can do a lot of strange things.  So that's...  But it exists. It's always nice to know that it exists.  Yeah.  So depending on what type of functionality that you have on your stationary phase,  you change your cell activity.  And phenyl and pentafluorofenyl, they're not just more polar,  they also have other types of cell activities.  Because of the benzene ring.  So the benzene ring here, that can create these van der Waals forces  that we talked about last time.  You had a molecule morphine, I think it was.  So you got kind of a dipole here that will attract other aromatic molecules.  So here you got the stationary phase with an analyte aromatic ring sitting on top.  So that will hold on to aromatic compounds even harder.  And also you have that dipole interaction in your pentafluorofenyl group,  which also gives you a different selectivity.  So if you got aromatic compounds with similar polarity to a non-aromatic,  you can actually change that selectivity by choosing a different type of reverse phase column.  Questions?  When we're talking about the stationary phases in normal phase,  again we have the amino, the diol, silica, and different amide functionalities sticking on that.  And it's very, very important that if you want to use one of these stationary phases for normal phase separation,  you don't put water anywhere near that.  Because once you do that, you ruin that effect.  You only use these very apolar solvents.  Because when you got the silica, all these different cross links that you saw in between,  they can be broken by water, they can be hydrolyzed, and that will change the chemistry of the particle.  So actually when you work here, we also have some small one-time use columns we can use to purify.  And in those, you have to dry them in an oven.  And you will also see working in the summer, which is very humid,  but they will not work as well as now where everything is dry.  So also some that makes these, they actually add a little bit of water,  so they know that it's 0.05% water, something like that.  Simply to say, we cannot control water, so we add a controlled amount.  But in general, we try to keep these as water-free as possible.  Yeah.  So.  This also why this chromatography is not as popular as the reverse phase,  because we have a reproducibility problem, because of moisture and all kinds of things.  Yeah.  So the difference, like I said, with normal phase and aqueous normal phase,  HILIC, is that when you use silica or amino as a HILIC phase,  you actually put water in there deliberately, because here you see that silica surface here.  And actually when you put water in, all that layer will be filled with water.  And that water layer is actually your stationary phase, or you can say, can you say that?  Yeah.  Yeah.  So it's the water layer that is in here that is important,  and not necessarily the functional groups itself.  So when you have used hydrophilic interaction chromatography,  you got an amino phase, and you got all that water bound in between the amino groups,  sticking out from the silica, which means that when your mobile phase has a high organic content,  so when you start out with acetonitrile, for example,  the polar compounds, they are not fond of acetonitrile,  they'll go in and hide here in the water layer.  So they'll stick to that water layer with the stationary phase,  and the mobile phase with high organic will just pass through and let them rest there for a while.  So when you want in HILIC, you want your compounds to leave the bar, you want them to leave the column,  you increase the water content, because then the compounds can diffuse out from the water layer into the mobile phase  and leave the column.  So here, this water layer is really essential for how that works.  So we use water, we use acetonitrile, we don't use alcohols,  because when you put alcohols in here, that will confuse everything and make a very weird layer in between,  and we have no HILIC effect at all.  So the water is the key part of this separation.  So now we're going to start up whole, hope that works.  So here you got a TLC plate, so it's called thin layer chromatography.  Have you heard about that before?  Yeah, very quiet.  So here you got, like you got a column, you got a flow of mobile phase passing through,  so the compounds move in this direction.  And then we got these acylated homocerin lactones, these are bacterial signal molecules,  bacteria that make them to communicate with each other and send war signals and signals to colonize the surface,  and there are a lot of different analogs of these.  And they all have this homocerin lactone head, and then they got an aliphatic chain here of different length.  So in this case you got a very long aliphatic chain, so you got a compound that is very apolar or polar.  Apolar, yeah, correct.  So this is very apolar.  This here on the other hand got a hydroxyl group, so it's polar.  So apolar to polar, eluding with 60% methanol in water.  What type of chromatography do we have here?  So go vote and see how it works out.  Click the bar on top.  Yeah, I couldn't see it.  I think it's good to have.  Yeah, I think it's good to have.  Hello.  Yeah.  Yeah.  Stopping the poll.  Anyone dares to tell me what they voted before I put on the distribution?  Yeah.  See if it works.  That's beautiful.  Almost 70% on reverse face.  A few on normal face and HILIC, but the correct answer is in fact reverse face.  So you got the most apolar compounds coming out first, and then you moved to the polar ones.  Both normal face and HILIC, they'll have the polar compounds coming out first.  And also if you're in doubt between normal face and HILIC, you can see that on the solvents because we added water, which is a no-no in normal face.  So this is the way that you can recognize these different kinds of chromatography.  This is also good for the ones who know what happened here is that we added the compounds, and actually then you draw the TLC plate, and if you don't draw it, then it doesn't work, because then it will kill the bacteria after that.  Anyway, then you have the TLC plate, and we then have a argon at 50 degrees.  When it's almost starting to solidify, and then you have a signal bacteria that can produce this blue color.  Then you put it in, you shake it very fast, and you put it on the TLC plate, and you wait for about four seconds.  Oh, yeah, three, four days, and then here you can simply see then it gets colored where you have the signaling molecule.  This is also an example of where we interface the chromatography with biology and use biology as a detector.  Yep.  Yep.  We'll move directly to a new poll.  Now I'll give you some hints on how you can recognize the different types, and I'm just going to start that.  So here again, we want to know what type of chromatography it is, but we will also, as a secondary question, you can vote two times.  You can ignore the second part of the question if you want to, but which of these diacetyl glucose and glucose is peak number one and peak number two?  So from the text up there, see if you can try to recognize the type of chromatography and then figure out whether or not the A-polar or the polar analog of glucose will come out first.  And if you don't see the question on your computer, try refreshing the browser. I don't know how quickly it updates.  And again, you can just ignore the second part of the question if you...  Okay.  Okay.  Okay.  Any final votes?  Last one coming in.  So, excellent.  So you all guessed more or less that this was HILIC. You definitely... none of you voted for reverse waste, which is great. You realize that you started with a high organic content, so which means that it's either normal phase or HILIC, but you got water, which means that it's HILIC.  And the order of these two, that is more difficult. So that's 50-50 on the votes. And this is because we both have polarity of the compounds and polarity of the stationary phase, and now everything is confused.  But HILIC, we got the A-polar compounds coming out first. And which of these two are the most A-polar one?  Come on.  So, in this case, you got extra C-groups. You got acetyl sitting instead of hydroxy, which makes it more A-polar.  So, this one will be the one coming out first, and this one will be the one coming out last.  And this would be the opposite if we had reverse space. So, now I understand why you're confused.  So, like we talked about, that we can predict these log D values. And log D predictions, they're good for prediction of retention time for some compounds.  So, you got these homo-saryon lactones that you got on the TLC plate before.  It's a beautiful linear relationship between retention time and log D value.  So, you see, the longer the chain gets, the higher the log D value, the more A-polar of the compound, the longer the compound will stick to a reverse phase column.  And recall here, this is actually calculated. This is not measured in a lab. This is just a calculated one.  But also here, even though, let's say, it actually doesn't calculate it right or a little bit wrong, but then, you know, adding here still helps a lot.  So, you know, it would only move it up. So, that's why it makes such a good correlation.  So, these calculation tools are very, usually very good in one to predict a small modification.  Yeah. So, actually, when you got a chromatogram like this, again, you can predict the order of retention based on polarity.  So, now you got a lot of exercises in the printout.  And I'm just going to go because I realize that the last exercise, this is the last exercise for this section.  So, there's coffee on the table. There's cake on the table. You can move to the next room.  And we'll give you what, half an hour, Christian?  Yeah.  Yeah. Half an hour to talk about the exercises and then we'll...  Yeah. Coffee and sugar and everything should be next door.
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