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 talked 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, but, but.

The first phase is usually the C18 chains. So they're sticking out from the particles and the interaction from your molecules to this is very, very fast. You know they're moving out here and then. And the reverse phase is the faster principle here. This is why it's usually the most efficient.

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 the answer. What is your? Yeah, and why is that? Because it has this big apolar part. Yeah, so it's got this long aliphatic chain which fits like a hand in a glove with the C18 that Christian showed 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 a charge either at low or high pH because amides, they're 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 apolar 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? I've heard that you got it. Yeah. And you also have the explanation you told me.

Yeah, 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. Yeah. 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 at 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, 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 chain 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. So this is the other end of the scale, and this is now we got the four remaining structures, this is where it gets difficult. What do you think that happens if we then remove a hydroxyl group here?

Will it come out before or after when it interacts with this? 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 V 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 isomeres you worked on.

Let's say one of the teams get this as the biosynthesis. Then you should know that if you add hydroxyl groups, they 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 you are 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 pentabromosutilin. We got five bromines. Bromines will contribute to a positive log V 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. High pH is a weak acid, 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.

And 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 V 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 the 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 enyl, which has the same effect as a phenyl. It's a weak acid. 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, is 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. Yeah. So a little bit of a difficult exercise, but hopefully,

you'll get a feeling of movements and relative polarity. There will be much more. So, when we're talking about separations based on polarity, we got free separation principles,

so three types of chromatography you're going to learn about. We got the reverse phase, normal phase, and helix, 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 got a very high polarity range.

So it can actually hold on to quite polar compounds and is 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 got the polar ones, they will come out first because the stationary phase that Christian 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 groups.

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 of 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. So, when we have 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 apolar 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 bead when you got the R group sticking out, no R group, just silica. So it's really good for fats and sterols and triglycerides and all these apolar compounds and the order of elution is the opposite.

So it's the most apolar compounds, they will be pushed quickly through 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, call that 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, ethyl acetate and methanol.

So these are the apolar 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 we'll 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 got normal phase the old, old fashioned way, this is the modern way and this is the really modern way. So, not a lot of people use HILIC and not a lot of people understand exactly how HILIC works.

It's one part chromatographic science 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 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 apolar 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, spitter 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 apolar solvents, heptane and all that we can't use for making ions. Oh, now this one's up. While in HILIC 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 the 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 HILIC is black magic, sometimes it's worth just looking at some of the more polar reverse phases. So that can be a phenyl, cyano or pentafluorophenyl 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 voodoo, so that can do a lot of strange things and so that's...

So, depending on what type of functionality that you have on your stationary phase, you change your selectivity. And phenyl and pentafluorophenyl, they're not just more polar, they also have other types of selectivities.

And that is 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 pentafluorophenyl 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, they will not work as well as now where everything is dry. So, also some that make these, they actually add a little bit of water so they know that it's 0.05% water, something like that. Simply to say, you cannot control water, so we add a controlled amount.

But in general, we try to keep these as water-free as possible. This is 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.

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. 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, here 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 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 we 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 a poll. 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 acetylated homoserine lactones. These are bacterial signal molecules. Bacteria, they 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 homoserine 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 eluting with 60% methanol in water.

What type of chromatography do we have here? So, go vote and see how it works out. The band off.

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 phase. A few on normal phase and hillock

but the correct answer is in fact reverse phase. So you got the most apolar compounds coming out first and then you move to the polar ones. Both normal phase and hillock, they'll have the polar compounds coming out first. And also if you were in doubt between normal phase and hillock

you could see that on the solvents because we added water which is a no-no in normal phase. 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 dry the GLC plate and if you don't dry it then it doesn't work because then it would kill the bacteria after that. Anyway, then you have the GLC plate and we then have an argor at 50 degrees. That's when it's almost starting to solidify. And then you have a signal of bacteria

that can produce this blue color and then you put it in, you shake it very fast and you pour it over the GLC plate and you wait for about four, six, oh yeah, three, four days and then here you can simply see then it gets colored and that's where you have the signaling molecules. This is also an example of where we interface your chromatography with biology

and use biology as a detector. Yeah, 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 so 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 apolar or the polar analog of glucose will come out first. 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...

Any final votes? That was more difficult maybe?

So... Excellent! So you all guessed more or less that this was HILIC? Definitely none of you voted for reverse phase, 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 apolar compounds coming out first. And which of these two are the most apolar one?

In this case you got extra C groups, you got acetyl sitting instead of hydroxy which makes it more apolar. So this one will be the one coming out first and this one will be the one coming out last. And it would be the opposite if we had reverse phase.

So now I understand why you were 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 here you got these homo-serine 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 more, the longer the chain gets,

the higher the log D value, the more apolar 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 usually very good and want to predict a small modification. 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 printer and I'm just going to go because I've realized 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? Half an hour to talk about the exercise

and then we'll, yep, coffee and sugar and everything should be next door.