Chromatographic Separation #1
Nielsen, Kristian
N. N.
Chromatographic separation; Chapters: 0:00 - Introduction;
 Good afternoon everyone. We're starting with a poll. I don't know many of you were traveling maybe last week. So this year we're going to try out some SMS voting and any of you not here last Monday. So it works that way that you lock on to the wireless internet with your smartphone or your computer and unfortunately people with no computers or smartphones cannot vote so they have to have their neighbor help them. Yeah, yeah we won't ask you yet. So but you lock on to this web page up here and then hopefully it will be so. I think you need the three W's and these have to be in capsule. Yeah, so try to lock on and these seven statements should appear on your phones or your computers. So we'll see if it's going to work out today. We have Cecil here from Learning Lab DTU seeing how these new teaching tools are working out and got video recording on today as well. So we're going to try out a lot of different things today. So yeah, try it. Okay, it's working. Take a couple of minutes. And again, there is more than one correct answer so vote as many times as you like. Excellent. Yeah. I'm going to buy a stove so you can get it for yourself. I'm going to buy a stove so you can get it for yourself. I'm going to buy a stove so you can get it for yourself. Excellent. It's working again today. So I'll stop this one. Maybe it's unfair to start with a quiz. But like we talked about yesterday, last Monday, this year we're going to try to use the learning objectives a little bit more actively than has been done the other years. So if you recall, one of the learning objectives from last week was to know the difference between primary and secondary metabolism. And all of these statements have something to do with the common misconceptions or conceptions about primary and secondary metabolism. So five of you voted that secondary metabolites can be found in oral living organisms. And that, in fact, is true. Organisms have different capabilities of producing molecules, but even we as humans produce secondary metabolites, you can say hormones are a sort of secondary metabolites. So that is a true statement. I'm very happy that none of you answered that they are essential for growth, development, and reproduction, because that is what the primary metabolites are involved with. Perhaps as humans they probably are because of the pheromones, but yeah. So yeah, very happy about that. A lot of you agreed that they're made from the intermediates that are coming from the primary metabolism, and that is correct as well. Secondary metabolism can be seen as a second layer of metabolism in that sense. Also very happy, confer a competitive advantage to the producing organism. And in that sense, we talked about last time they're not essential in for growing and living. But they're essential for growing and living better than others and staying alive. Then a few of you voted that they include sugars, fats, and nucleic acids. And this is very interesting because this is not neither true or wrong, because we have something of a gray area, in particular with the sugars and fatty acids, not fats, but fatty acids. And that is very much in the gray area between primary and secondary metabolism. Are produced under all growth conditions? None of you voted for that? Excellent, that is true. Secondary metabolism is very much dependent on the growth conditions, and are the same in all members of the same species? None of you thought that either? That is also correct because it's actually a way that we can differentiate different species. Excellent, well done. You fulfilled one of the first learning objectives of this course. So today it's going to be chemistry day. You're all excited about that. So we're going to start talking about chromatographic separation of natural products, secondary metabolism. And one of some of the things that we're going to teach you today is to talk about liquid chromatography. Christian gave a quick overview last time, but today we'll go more into the theory behind it. And Christian will explain to you how the different components of an HPLC works. Also we'll talk a lot more about functional group chemistry. Last time you had a little recap on functional groups because natural products are particularly rich in functional groups, and this is exactly what we exploit in chromatography. And then we'll talk in particularly about silica as a stationary phase and reverse phase separation because this is one of the things that you're really going to learn a lot about in this course. I'm going to ask you about for your exams and you're going to use it for your exercises. So hopefully by the end of today you'll be able to outline the main mechanisms that we use to separate compounds. You'll be able to highlight the different types of chromatography. Again here in particular reverse phase separations, but also something called helic and normal phase separations and explain when are we going to use them, for what type of compounds are they particularly useful. And then you're going to do a lot of prediction. Just from looking at structures and molecules, how would you predict and expect them to behave in a chromatographic system? So we'll have four parts today and it's going to be a little bit of mix and match. So we start out by talking about functional group chemistry and then you're actually going to have some group exercises. You can move around, move to the next room because we realized that last time this is a very small room with very little oxygen in it. So move to the next room. It has oxygen and it will have coffee and cake as well. So yeah and then Christian will take over also have some lectures and some exercises in between. So hopefully it will break up the day and not be too tiresome. So some of the things that we talked about last time is what we have a plate of microorganisms that are capable of producing a lot of different molecules. How, which steps would we go through in order to figure out what they're capable of doing? And that goes for both known compounds that could be toxins or it could be completely new molecules that we want to use for new drugs. And even though we have a slightly different approach depending on whether or not we know what we're looking for or we have absolutely no clue what we're looking for, we go through some of the same steps in the analysis. And you came up with some great suggestions. And the essence of those steps that you're all listed were extraction, separation, detection, identification. And today we're going to talk in particular about that part here in the middle, separation but also a little bit about detection. And then I'm very fond of that statement up in the corner here saying isolation, purification, separation, identification and celebration because that is exactly what this is about. When you actually manage to go through these steps, we have a party. So when it comes to separating compounds, what chemical characteristics can we actually exploit? Do you have any ideas for that? If you have these three different compounds, how are they different? How can you separate them based on their differences? No? We have three major chemical or physical characteristics that we use. First of all, we can use their polarity. How soluble are they in water? So that is a major characteristic. Any suggestions other than that? Yeah? Exactly. So some compounds can differ quite a lot in size. And here again, of course, we see the two-dimensional structure. Size will play an even bigger factor when we've got the three-dimensional structure because some compounds can fold in and make globular shapes and whatever. So size is a really good way of separating compounds as well. Any other than these two? If you got functional groups like this, some of them are capable of carrying a charge. So that is the last major thing that we can separate compounds based on. If we're talking about compounds that we know that have a specific biological function, we can also use affinity. So we can separate compounds based on whether or not they will bind to a specific protein or receptor. But that is not something that goes for all molecules. That is not something that we can use as a general separation principle. So in this course, we'll go through the three top ones here. And the reason why we want to know all the mechanisms that we can use is that sometimes compounds do not differ a lot when it's just looking at one characteristic. So for example, you can have compound A and compound B, which are very similar in their polarity. So no matter how much we optimize the different solvents and how we will never be able to separate these two. However, so we introduce second dimension, the size, and the third dimension, the charge. And in particular, the charge, whether or not something have a positive or a negative charge, that is really something quite distinctive of a compound. And this is what we call orthogonal separation. Because in theory, these characteristics does not have anything to do with each other. That is not entirely true. Because the polarity will also depend on the size of the compound. Usually a very big molecule, if all functional groups are the same, that is more A polar than a very small molecule. And also the charge can change the polarity of a compound. But I'll return to that. So when we're talking about natural products, exploiting functionalities is really important. We want to make the best of what we know about these compounds. We don't know a lot, but we know they have a lot of functional groups. And in fact, if you make statistics about all microbial metabolites that are known today, you can see this distribution. You can see that if you look at compounds from filamentous fungi and bacteria, and you should see the total number as well, that also includes algae and other types of microorganisms, you can see about half of them, they have an acidic functionality. That could be an enol or phenol that is capable of exchanging a proton or simply a carboxylic acid. And the carboxylic acids are about half of the acidic functionalities that are present. Then amines, they can be positively charged, they can act as basis, not if they're close up in a ring or in an amide, but otherwise they can carry a charge. And you can see here about 28% of all bacterial metabolites, they have a cationic charge. So this is really, if you want to sort out and separate out compounds, you can remove almost a third of these compounds just by looking at a positive charge. So these are the functional groups that we talked about last time. And we try to exploit these functionalities when we look at chromatography. So Christians showed you this slide last time as well. This is a picture of some of the beads that we have in the stationary phase of chromatography. So this is what our compounds will bind to when we try to separate them out. So by packing a column with a lot of these different particles or beads, you can tell that if you change the selectivity, if you change the affinity towards specific functional groups, you can actually change how fast the compound can move through a column. So this is how a particle looks on the inside. So when we're talking about reverse phase chromatography, but also normal phase and hydrophilic interaction chromatography that we'll discuss today, the base of the particle is silica. So we have a lot of different cross links within the particle. And then we can have different changes to that silica particle. So we can introduce, here we've got a long C18 chain. And this is the basis of most reverse phase separation, that is C18. So it's a long aliphatic chain, which is very apolar. So that will attract very apolar molecules. So compounds with very polar functionalities, hydroxyl groups, amines, whatever, they will usually be repelled by this C18 chain. But in principle, it can be anything that you can change here, the R group. It can be hexyl, phenyl, amino groups, cyanogrups, diol groups. You can put everything on that will make your compounds stick to it. So you can change your selectivity towards specific compounds if you change the chemistry of the particle. So how many of you have heard about chromatography before, except for last time? Now you had a few. So I'll show you the very cartoonish explanation for the rest of you. The principle behind chromatography here again is that you have a stationary face. This should reflect the particles that you saw in the other picture as well. You got the round beads, which are functionalized with different types of R groups. So here is C18. And Christian mentioned the POP-CROLE model of chromatography last time. These are the bouncers. So these are the ones that decide who gets in and who gets out of the bars and how long it's going to take. So you have a mixture of analytes up here, and they will move through the column with a mobile face. So everything in chromatography is about are the compounds most fond of the stationary face or the mobile face? And the mobile face is moving at all times. So you've got a flow coming through here. So you'll have a constant flow of mobile face, which can attract your analyte molecules, or they can be absorbed into the stationary face. And then you can see that depending on the functionalities of your analytes, they will move faster, slow through the column. They will bind weakly or strongly to the stationary face. So maybe you're already guessed by now that the stationary face, the particles that make up the stationary face, they are really the essential part of chromatography. This is where we can make all the difference. This is where we can buy different columns and obtain different kinds of separation. So here we have two different kinds of particles. And you can see that they differ a little bit in their structure. And actually, there's so much different from brand to brand that you can get selectivity just from switching to another vendor than changing the stationary face. Even though they're both C18. So even though you've got the same R group sitting on there, the way that the particle is constructed will actually sometimes matter even more. So when you will read papers about how compounds are separated and you say that they, you can read that they use the particular brand, Phenomenics, something column, you can't just go out and buy a different brand and expect exactly the same separation. So in fact, when you got the picture before, you got the compounds passing through the column, they will not just interact with the surface of the particle. In fact, you'll see the compounds moving into the particle. They'll be absorbed into the particle. And we got this porous layer, older columns. They have a particle that is completely porous, which means. Like sponge, you know, in the bathroom. Yeah. So, so in fact, that's the compounds can pass all the way through the particle. And that of course takes time, which makes sometimes very unpredictable how compounds will behave with a certain particle. But today, most modern particles, they have like a core, a heart core shell, and then they have a porous shell on the outside. And the particle size is then you got, in this case, you got a 0.35 micron porous shell. Here you got 0.23 porous shell. So that the compounds from the analyte will only move through the porous layer, which means that it will go faster and you can get better separation. So again, if you buy a new column with C18 that is constructed this way, you will have a much different type of selectivity, a much different type of chromatography compared to an old C18 type particle. So the chromatographic mechanisms that will go through in this course, they're all mainly based on separation based on polarity. And we have the scale compounds going from polar to a polar. And how polar or a polar a compound is will very much depend on the functional groups, like I said. So right here in the very polar end of the scale, you got salts. So if you got compounds with a permanent charge and you got a counter ion sitting there, these will be very, very polar. You got different types of acids, carboxylic acids are the most polar ones, but then you got alcohol groups, ketones, ethers, halogens, aromatics, aliphatic compounds, and then fluorinated compounds all the way out to the end. These are usually the ones that you'll encounter in this course. And maybe some of you remember from last time, we had a pole with a natural compound and a synthetic derivative. And the synthetic derivative had floor stuck onto the structure. So this was a strategy for the pharma company to increase the polarity of that compound. So by introducing different functional groups, you can change the polarity of a compound. The scale that we use to assess how polar or non-polar compounds are is called LOC D. And it's a logarithmic value. So you see the distribution of compounds between water and octanol. So of course, the octanol is in the top one. So the lower the number, the more polar, the more of the compound will be in the water phase. So that means that you can actually have negative values. So if all of the compound is, will be in the water phase, you'll have a negative value as well. Here you can see the effect on different functional groups on the LOC D value. And this is just an assessment. Modules will not always behave as linear as you would expect them to do. Some of them do. But it's a good, gives you a good feeling of how the different functional groups will affect the LOC D value. So if you got a hydrogen and you exchange that with the hydroxyl groups, you'll have a change of minus 0.67. So on the other hand, if you introduce an aromatic group, you'll see an increase of 2.13. So you can actually predict sometimes how the polarity will be of different natural compounds. So here you got canamison, an antibiotic made of free rings of sugar, very high of the sire to be in the water phase. So it will have a negative value of 12.6. Then we got another antibiotic, TDA, trebotithiac acid, also very polar compound. Just for comparison, we'll put ethanol in there maybe. That is a better indication on the scales that you're used to. Then we got a mycotoxin here, which is medium polar. The condom, the spice, the chili, the compound that will give you that burning sensation. This is the quiet A-polar compound. This is one of the explanations why it helps to drink milk. A lot of fatty things, drink a bowl of fat and then you won't feel the spiciness. Then also a toxin, also very A-polar, and then you got bicaratine, which is all the way in the other end of the scale, very aliphatic molecule. So it's very, very A-polar. But I mentioned in the beginning that these free principles that we got to separate compounds, they're not completely orthogonal. That is because that pH can affect the polarity of the compound. That is because compounds with charges, they have pKa values. They can change the charge depending on what the pH they're in. You had TDA before, which was a very polar molecule. In fact, if you change pH, it has a pKa value of 3.7. So at high pH above 3.7, it will lose that proton sitting here in the carboxylic acid. This will give it a negative charge, which will make it very soluble in water. On the other hand, if you decrease pH, you give it a lot of proton H+, you make sure that the carboxylic acid is protonated at all times, which will make it more soluble in organic solvents. It will make it more A-polar. Make sense? Yep. Again, here we've got Rogfortzen. It got an amine sitting there. It can carry a positive charge. It will do that at low pH, so if you've got a lot of H+, this will be N+, which makes it very soluble in water. Again, if you increase pH, it will be deprotonated and more A-polar. So if you want to extract this from water, we'll easily acetate or hexane or something like that, and you have to be at high pH. Here, we have to go low. Yeah. Exactly. By knowing this about your compounds, this is a very selective factor that you can use. Then, of course, you've got ethanol. It does not have a charge at any pH, so you have a constant lockdown value. It will not change. It will actually up here, but it can't calculate it. It will start to make a negative charge, but at the extreme, it is in concentrated sodium hydroxide. Yeah. But at natural pH values, it will never be charged. We're talking about C-lock B value, which is a pH-adjusted value. One thing, this is a calculation tool we have. This is not experimental data, but we have computer programs. We actually have a campus license, so this we can just calculate. It may not be totally true, but it's going to be pretty close to... Yeah. To what's right. Yeah. So, using something like the table that we showed before, that you will see an effect of something-something. It's based on some experiments, but some compounds are better predicted than others. Rural foam, acids will be more A-polar at low pH, and bases will be more A-polar at high pH. You got the scale again from polar to A-polar, and why are we interested at all in knowing why our compounds... If our compounds are polar or A-polar, that is because this defines the type of chromatography that we're going to use. So you'll see the big hat up here, that is reverse face, and that covers actually 80% of the polarity range, something like that. It will cover most natural products. So this is why you have to spend so much time on learning about reverse face chromatography, because this is actually the most versatile and the most commonly used method. But you're also going to learn about normal face separation, which is in the A-polar end of the scale, and you're going to learn about hydrophilic interaction chromatography, which is other end of the scale. Using the extremely polar compounds like the sugars, canamysin that I showed you before. And everything inside the cell, if you recall you by chemistry, you would see that actually all the things in the TCA cycle, all the phosphorylated compounds, I mean, they're down here, and if you inject them on a reverse face column, just going to pass directly through. So if you're ever going to look at primary metabolites, that is definitely in this end of the scale. Then, not today, but maybe later. Haven't decided yet. You're going to learn about other types of chromatography, ion pair and ion exchange, but very briefly. But again, it's important to know which type of column would you pick out from the shelf if you want to analyze a particular type of compound. Yeah, so special emphasis in reverse face, and then something about normal phase in hydrophilic interaction chromatography, but most importantly, that you will be able to know when to expect reverse face to fail, and when do we have to go dig somewhere else on the shelf for something other than that. So also talking about polarity and chromatography, this is very big part of that is the mobile phase, the solvents. But we use solvents for a lot of different things. We use the solvents for extraction. We use them as part of the HPLC system. We flush solvents through all of detectives, and we actually use solvents when we want to run bioassays, for example. And solvents are very important, and even though that you're not chemists, it's very important to know what type of compound that you're dealing with and what solvents that you can use. Because one of, in our many collaborations, one of the biggest questions that will arise every time, we as chemists have produced a compound that we want to test in an antibacterial assay, and then the people running the assay, they will come and ask, what should we dissolve it in? And we will like you, for people that most likely will be the one running the assays, to know what solvent to put your things in. And sometimes things are not always compatible, because we have certain requirements for the solvents that we're going to use to dissolve or sample in. So maybe we have a compound that is not soluble in water at all, so we need to use a more apolar solvent. But when we start to analyze it with the HPLC, we also want to make sure that it's actually mixable with the solvents that we have in our system, that it's not making two-phase systems, that you got your sample floating on top of your mobile phase running through, then you won't have any of that fantastic functional group chemistry, because it will be isolated in a little bubble. Then we have something called illotropic strength, which is the power of a solvent to take the molecules with them in the mobile phase and take them through the column. We also have to consider viscosity, because that will influence the pressure of a chromatographic system. Then when we are passing them through the detector, we also have to know whether or not the solvent itself will affect the detection. So if we got a solvent that has great UV absorption, it doesn't matter how good our natural compounds are at absorbing light, because they won't see them at all. We have a huge background peak. That's why we don't use acetone, because that takes all the light up to 350 nanometers, and that's where we see most of the goodies. So this is extremely important, but we also have some solvents that will cause a lot of different annoying background ions and MS as well. And then again, the last step, some solvents are excellent antibiotics. Some compounds have an effect on immunosystems and whatever, so sometimes you have some great, great positive results in your acid, just because you picked the wrong solvent. So all of these things are very important, but the first thing to consider is the relative polarity of these solvents. So again, further down in the polar end, we got water as the most polar solvent, then we're moving up to methanol or other alcohols for that matter. That could be butanol, probenol, ethanol, the whole thing, acetonitrile, then we're moving into the apolar solvent. So this is where we got ethyl acetate, dichloromethane, and heptane. So we got the full range of solvents, and we got the full range of polarity in our compounds, so we just have to mix and match what fits together, and which will fulfill the above requirements that we have. So it's very easy. So we'll start with a little exercise and have a break as well after that. Oh, after four or again. Yeah, so we have printed out the exercises here. So what we want you to do is to try to rank these natural compounds according to their polarity, both at high pH and low pH. This is where polarity changed with pH for certain compounds. So you have to identify the potential functional groups that can be charged at different pHs. And then we'll go through the exercise afterwards. How long should we take, Christian? 15 minutes? Yeah, you can show this one and the next one. So we take the break. You need the break now? Yeah, so you can have the exercise and the break together. Yeah, in 20 minutes, yeah. So at two o'clock we'll... There are many exercises here. So I can't... I'm sorry, okay, there's only one with this one. Yeah. So 15 minutes, yeah. 15 minutes, yeah.
 Good afternoon everyone. We're starting with a poll. I don't know many of you were traveling  maybe last week. So this year we're going to try out some SMS voting and any of you  not here last Monday. So it works that way that you lock on to the wireless internet  with your smartphone or your computer and unfortunately people with no computers or  smartphones cannot vote so they have to have their neighbor help them. Yeah, yeah we won't  ask you yet. So but you lock on to this web page up here and then hopefully it will be  so. I think you need the three W's and these have to be in capsule. Yeah, so try to lock  on and these seven statements should appear on your phones or your computers. So we'll  see if it's going to work out today. We have Cecil here from Learning Lab DTU seeing how  these new teaching tools are working out and got video recording on today as well. So we're  going to try out a lot of different things today. So yeah, try it. Okay, it's working.  Take a couple of minutes. And again, there is more than one correct answer so vote as  many times as you like. Excellent.  Yeah.  I'm going to buy a stove so you can get it for yourself.  I'm going to buy a stove so you can get it for yourself.  I'm going to buy a stove so you can get it for yourself.  Excellent.  It's working again today.  So I'll stop this one.  Maybe it's unfair to start with a quiz.  But like we talked about yesterday, last Monday, this year we're going to try to use the learning  objectives a little bit more actively than has been done the other years.  So if you recall, one of the learning objectives from last week was to know the difference  between primary and secondary metabolism.  And all of these statements have something to do with the common misconceptions or conceptions  about primary and secondary metabolism.  So five of you voted that secondary metabolites can be found in oral living organisms.  And that, in fact, is true.  Organisms have different capabilities of producing molecules, but even we as humans  produce secondary metabolites, you can say hormones are a sort of secondary metabolites.  So that is a true statement.  I'm very happy that none of you answered that they are essential for growth, development,  and reproduction, because that is what the primary metabolites are involved with.  Perhaps as humans they probably are because of the pheromones, but yeah.  So yeah, very happy about that.  A lot of you agreed that they're made from the intermediates that are coming from the  primary metabolism, and that is correct as well.  Secondary metabolism can be seen as a second layer of metabolism in that sense.  Also very happy, confer a competitive advantage to the producing organism.  And in that sense, we talked about last time they're not essential in for growing and living.  But they're essential for growing and living better than others and staying alive.  Then a few of you voted that they include sugars, fats, and nucleic acids.  And this is very interesting because this is not neither true or wrong, because we have  something of a gray area, in particular with the sugars and fatty acids, not fats, but  fatty acids.  And that is very much in the gray area between primary and secondary metabolism.  Are produced under all growth conditions?  None of you voted for that?  Excellent, that is true.  Secondary metabolism is very much dependent on the growth conditions, and are the same  in all members of the same species?  None of you thought that either?  That is also correct because it's actually a way that we can differentiate different  species.  Excellent, well done.  You fulfilled one of the first learning objectives of this course.  So today it's going to be chemistry day.  You're all excited about that.  So we're going to start talking about chromatographic separation of natural products, secondary metabolism.  And one of some of the things that we're going to teach you today is to talk about liquid  chromatography.  Christian gave a quick overview last time, but today we'll go more into the theory behind  it.  And Christian will explain to you how the different components of an HPLC works.  Also we'll talk a lot more about functional group chemistry.  Last time you had a little recap on functional groups because natural products are particularly  rich in functional groups, and this is exactly what we exploit in chromatography.  And then we'll talk in particularly about silica as a stationary phase and reverse phase separation  because this is one of the things that you're really going to learn a lot about in this  course.  I'm going to ask you about for your exams and you're going to use it for your exercises.  So hopefully by the end of today you'll be able to outline the main mechanisms that we  use to separate compounds.  You'll be able to highlight the different types of chromatography.  Again here in particular reverse phase separations, but also something called helic and normal  phase separations and explain when are we going to use them, for what type of compounds  are they particularly useful.  And then you're going to do a lot of prediction.  Just from looking at structures and molecules, how would you predict and expect them to behave  in a chromatographic system?  So we'll have four parts today and it's going to be a little bit of mix and match.  So we start out by talking about functional group chemistry and then you're actually going  to have some group exercises.  You can move around, move to the next room because we realized that last time this is  a very small room with very little oxygen in it.  So move to the next room.  It has oxygen and it will have coffee and cake as well.  So yeah and then Christian will take over also have some lectures and some exercises  in between.  So hopefully it will break up the day and not be too tiresome.  So some of the things that we talked about last time is what we have a plate of microorganisms  that are capable of producing a lot of different molecules.  How, which steps would we go through in order to figure out what they're capable of doing?  And that goes for both known compounds that could be toxins or it could be completely  new molecules that we want to use for new drugs.  And even though we have a slightly different approach depending on whether or not we know  what we're looking for or we have absolutely no clue what we're looking for, we go through  some of the same steps in the analysis.  And you came up with some great suggestions.  And the essence of those steps that you're all listed were extraction, separation, detection,  identification.  And today we're going to talk in particular about that part here in the middle, separation  but also a little bit about detection.  And then I'm very fond of that statement up in the corner here saying isolation, purification,  separation, identification and celebration because that is exactly what this is about.  When you actually manage to go through these steps, we have a party.  So when it comes to separating compounds, what chemical characteristics can we actually  exploit?  Do you have any ideas for that?  If you have these three different compounds, how are they different?  How can you separate them based on their differences?  No?  We have three major chemical or physical characteristics that we use.  First of all, we can use their polarity.  How soluble are they in water?  So that is a major characteristic.  Any suggestions other than that?  Yeah?  Exactly.  So some compounds can differ quite a lot in size.  And here again, of course, we see the two-dimensional structure.  Size will play an even bigger factor when we've got the three-dimensional structure  because some compounds can fold in and make globular shapes and whatever.  So size is a really good way of separating compounds as well.  Any other than these two?  If you got functional groups like this, some of them are capable of carrying a charge.  So that is the last major thing that we can separate compounds based on.  If we're talking about compounds that we know that have a specific biological function,  we can also use affinity.  So we can separate compounds based on whether or not they will bind to a specific protein  or receptor.  But that is not something that goes for all molecules.  That is not something that we can use as a general separation principle.  So in this course, we'll go through the three top ones here.  And the reason why we want to know all the mechanisms that we can use is that sometimes  compounds do not differ a lot when it's just looking at one characteristic.  So for example, you can have compound A and compound B, which are very similar in their  polarity.  So no matter how much we optimize the different solvents and how we will never be able to  separate these two.  However, so we introduce second dimension, the size, and the third dimension, the charge.  And in particular, the charge, whether or not something have a positive or a negative  charge, that is really something quite distinctive of a compound.  And this is what we call orthogonal separation.  Because in theory, these characteristics does not have anything to do with each other.  That is not entirely true.  Because the polarity will also depend on the size of the compound.  Usually a very big molecule, if all functional groups are the same, that is more A polar  than a very small molecule.  And also the charge can change the polarity of a compound.  But I'll return to that.  So when we're talking about natural products, exploiting functionalities is really important.  We want to make the best of what we know about these compounds.  We don't know a lot, but we know they have a lot of functional groups.  And in fact, if you make statistics about all microbial metabolites that are known  today, you can see this distribution.  You can see that if you look at compounds from filamentous fungi and bacteria, and you  should see the total number as well, that also includes algae and other types of microorganisms,  you can see about half of them, they have an acidic functionality.  That could be an enol or phenol that is capable of exchanging a proton or simply a carboxylic  acid.  And the carboxylic acids are about half of the acidic functionalities that are present.  Then amines, they can be positively charged, they can act as basis, not if they're close  up in a ring or in an amide, but otherwise they can carry a charge.  And you can see here about 28% of all bacterial metabolites, they have a cationic charge.  So this is really, if you want to sort out and separate out compounds, you can remove  almost a third of these compounds just by looking at a positive charge.  So these are the functional groups that we talked about last time.  And we try to exploit these functionalities when we look at chromatography.  So Christians showed you this slide last time as well.  This is a picture of some of the beads that we have in the stationary phase of chromatography.  So this is what our compounds will bind to when we try to separate them out.  So by packing a column with a lot of these different particles or beads, you can tell  that if you change the selectivity, if you change the affinity towards specific functional  groups, you can actually change how fast the compound can move through a column.  So this is how a particle looks on the inside.  So when we're talking about reverse phase chromatography, but also normal phase and  hydrophilic interaction chromatography that we'll discuss today, the base of the particle  is silica.  So we have a lot of different cross links within the particle.  And then we can have different changes to that silica particle.  So we can introduce, here we've got a long C18 chain.  And this is the basis of most reverse phase separation, that is C18.  So it's a long aliphatic chain, which is very apolar.  So that will attract very apolar molecules.  So compounds with very polar functionalities, hydroxyl groups, amines, whatever, they will  usually be repelled by this C18 chain.  But in principle, it can be anything that you can change here, the R group.  It can be hexyl, phenyl, amino groups, cyanogrups, diol groups.  You can put everything on that will make your compounds stick to it.  So you can change your selectivity towards specific compounds if you change the chemistry  of the particle.  So how many of you have heard about chromatography before, except for last time?  Now you had a few.  So I'll show you the very cartoonish explanation for the rest of you.  The principle behind chromatography here again is that you have a stationary face.  This should reflect the particles that you saw in the other picture as well.  You got the round beads, which are functionalized with different types of R groups.  So here is C18.  And Christian mentioned the POP-CROLE model of chromatography last time.  These are the bouncers.  So these are the ones that decide who gets in and who gets out of the bars and how long  it's going to take.  So you have a mixture of analytes up here, and they will move through the column with  a mobile face.  So everything in chromatography is about are the compounds most fond of the stationary  face or the mobile face?  And the mobile face is moving at all times.  So you've got a flow coming through here.  So you'll have a constant flow of mobile face, which can attract your analyte molecules,  or they can be absorbed into the stationary face.  And then you can see that depending on the functionalities of your analytes, they will  move faster, slow through the column.  They will bind weakly or strongly to the stationary face.  So maybe you're already guessed by now that the stationary face, the particles that make  up the stationary face, they are really the essential part of chromatography.  This is where we can make all the difference.  This is where we can buy different columns and obtain different kinds of separation.  So here we have two different kinds of particles.  And you can see that they differ a little bit in their structure.  And actually, there's so much different from brand to brand that you can get selectivity  just from switching to another vendor than changing the stationary face.  Even though they're both C18.  So even though you've got the same R group sitting on there, the way that the particle  is constructed will actually sometimes matter even more.  So when you will read papers about how compounds are separated and you say that they, you can  read that they use the particular brand, Phenomenics, something column, you can't just go out and  buy a different brand and expect exactly the same separation.  So in fact, when you got the picture before, you got the compounds passing through the column,  they will not just interact with the surface of the particle.  In fact, you'll see the compounds moving into the particle.  They'll be absorbed into the particle.  And we got this porous layer, older columns.  They have a particle that is completely porous, which means.  Like sponge, you know, in the bathroom.  Yeah.  So, so in fact, that's the compounds can pass all the way through the particle.  And that of course takes time, which makes sometimes very unpredictable how compounds  will behave with a certain particle.  But today, most modern particles, they have like a core, a heart core shell, and then they  have a porous shell on the outside.  And the particle size is then you got, in this case, you got a 0.35 micron porous shell.  Here you got 0.23 porous shell.  So that the compounds from the analyte will only move through the porous layer, which  means that it will go faster and you can get better separation.  So again, if you buy a new column with C18 that is constructed this way, you will have  a much different type of selectivity, a much different type of chromatography compared to  an old C18 type particle.  So the chromatographic mechanisms that will go through in this course, they're all mainly  based on separation based on polarity.  And we have the scale compounds going from polar to a polar.  And how polar or a polar a compound is will very much depend on the functional groups,  like I said.  So right here in the very polar end of the scale, you got salts.  So if you got compounds with a permanent charge and you got a counter ion sitting there, these  will be very, very polar.  You got different types of acids, carboxylic acids are the most polar ones, but then you  got alcohol groups, ketones, ethers, halogens, aromatics, aliphatic compounds, and then fluorinated  compounds all the way out to the end.  These are usually the ones that you'll encounter in this course.  And maybe some of you remember from last time, we had a pole with a natural compound and  a synthetic derivative.  And the synthetic derivative had floor stuck onto the structure.  So this was a strategy for the pharma company to increase the polarity of that compound.  So by introducing different functional groups, you can change the polarity of a compound.  The scale that we use to assess how polar or non-polar compounds are is called LOC D.  And it's a logarithmic value.  So you see the distribution of compounds between water and octanol.  So of course, the octanol is in the top one.  So the lower the number, the more polar, the more of the compound will be in the water  phase.  So that means that you can actually have negative values.  So if all of the compound is, will be in the water phase, you'll have a negative value  as well.  Here you can see the effect on different functional groups on the LOC D value.  And this is just an assessment.  Modules will not always behave as linear as you would expect them to do.  Some of them do.  But it's a good, gives you a good feeling of how the different functional groups will  affect the LOC D value.  So if you got a hydrogen and you exchange that with the hydroxyl groups, you'll have  a change of minus 0.67.  So on the other hand, if you introduce an aromatic group, you'll see an increase of  2.13.  So you can actually predict sometimes how the polarity will be of different natural compounds.  So here you got canamison, an antibiotic made of free rings of sugar, very high of the  sire to be in the water phase.  So it will have a negative value of 12.6.  Then we got another antibiotic, TDA, trebotithiac acid, also very polar compound.  Just for comparison, we'll put ethanol in there maybe.  That is a better indication on the scales that you're used to.  Then we got a mycotoxin here, which is medium polar.  The condom, the spice, the chili, the compound that will give you that burning sensation.  This is the quiet A-polar compound.  This is one of the explanations why it helps to drink milk.  A lot of fatty things, drink a bowl of fat and then you won't feel the spiciness.  Then also a toxin, also very A-polar, and then you got bicaratine, which is all the  way in the other end of the scale, very aliphatic molecule.  So it's very, very A-polar.  But I mentioned in the beginning that these free principles that we got to separate compounds,  they're not completely orthogonal.  That is because that pH can affect the polarity of the compound.  That is because compounds with charges, they have pKa values.  They can change the charge depending on what the pH they're in.  You had TDA before, which was a very polar molecule.  In fact, if you change pH, it has a pKa value of 3.7.  So at high pH above 3.7, it will lose that proton sitting here in the carboxylic acid.  This will give it a negative charge, which will make it very soluble in water.  On the other hand, if you decrease pH, you give it a lot of proton H+, you make sure  that the carboxylic acid is protonated at all times, which will make it more soluble  in organic solvents.  It will make it more A-polar.  Make sense?  Yep.  Again, here we've got Rogfortzen.  It got an amine sitting there.  It can carry a positive charge.  It will do that at low pH, so if you've got a lot of H+, this will be N+, which makes  it very soluble in water.  Again, if you increase pH, it will be deprotonated and more A-polar.  So if you want to extract this from water, we'll easily acetate or hexane or something  like that, and you have to be at high pH.  Here, we have to go low.  Yeah.  Exactly.  By knowing this about your compounds, this is a very selective factor that you can use.  Then, of course, you've got ethanol.  It does not have a charge at any pH, so you have a constant lockdown value.  It will not change.  It will actually up here, but it can't calculate it.  It will start to make a negative charge, but at the extreme, it is in concentrated sodium  hydroxide.  Yeah.  But at natural pH values, it will never be charged.  We're talking about C-lock B value, which is a pH-adjusted value.  One thing, this is a calculation tool we have.  This is not experimental data, but we have computer programs.  We actually have a campus license, so this we can just calculate.  It may not be totally true, but it's going to be pretty close to...  Yeah.  To what's right.  Yeah.  So, using something like the table that we showed before, that you will see an effect  of something-something.  It's based on some experiments, but some compounds are better predicted than others.  Rural foam, acids will be more A-polar at low pH, and bases will be more A-polar at high  pH.  You got the scale again from polar to A-polar, and why are we interested at all in knowing  why our compounds...  If our compounds are polar or A-polar, that is because this defines the type of chromatography  that we're going to use.  So you'll see the big hat up here, that is reverse face, and that covers actually 80%  of the polarity range, something like that.  It will cover most natural products.  So this is why you have to spend so much time on learning about reverse face chromatography,  because this is actually the most versatile and the most commonly used method.  But you're also going to learn about normal face separation, which is in the A-polar end  of the scale, and you're going to learn about hydrophilic interaction chromatography, which  is other end of the scale.  Using the extremely polar compounds like the sugars, canamysin that I showed you before.  And everything inside the cell, if you recall you by chemistry, you would see that actually  all the things in the TCA cycle, all the phosphorylated compounds, I mean, they're down here, and if  you inject them on a reverse face column, just going to pass directly through.  So if you're ever going to look at primary metabolites, that is definitely in this end  of the scale.  Then, not today, but maybe later.  Haven't decided yet.  You're going to learn about other types of chromatography, ion pair and ion exchange,  but very briefly.  But again, it's important to know which type of column would you pick out from the shelf  if you want to analyze a particular type of compound.  Yeah, so special emphasis in reverse face, and then something about normal phase in hydrophilic  interaction chromatography, but most importantly, that you will be able to know when to expect  reverse face to fail, and when do we have to go dig somewhere else on the shelf for  something other than that.  So also talking about polarity and chromatography, this is very big part of that is the mobile  phase, the solvents.  But we use solvents for a lot of different things.  We use the solvents for extraction.  We use them as part of the HPLC system.  We flush solvents through all of detectives, and we actually use solvents when we want  to run bioassays, for example.  And solvents are very important, and even though that you're not chemists, it's very  important to know what type of compound that you're dealing with and what solvents that  you can use.  Because one of, in our many collaborations, one of the biggest questions that will arise  every time, we as chemists have produced a compound that we want to test in an antibacterial  assay, and then the people running the assay, they will come and ask, what should we dissolve  it in?  And we will like you, for people that most likely will be the one running the assays,  to know what solvent to put your things in.  And sometimes things are not always compatible, because we have certain requirements for the  solvents that we're going to use to dissolve or sample in.  So maybe we have a compound that is not soluble in water at all, so we need to use a more apolar  solvent.  But when we start to analyze it with the HPLC, we also want to make sure that it's actually  mixable with the solvents that we have in our system, that it's not making two-phase  systems, that you got your sample floating on top of your mobile phase running through,  then you won't have any of that fantastic functional group chemistry, because it will  be isolated in a little bubble.  Then we have something called illotropic strength, which is the power of a solvent to take the  molecules with them in the mobile phase and take them through the column.  We also have to consider viscosity, because that will influence the pressure of a chromatographic  system.  Then when we are passing them through the detector, we also have to know whether or not  the solvent itself will affect the detection.  So if we got a solvent that has great UV absorption, it doesn't matter how good our natural compounds  are at absorbing light, because they won't see them at all.  We have a huge background peak.  That's why we don't use acetone, because that takes all the light up to 350 nanometers,  and that's where we see most of the goodies.  So this is extremely important, but we also have some solvents that will cause a lot of  different annoying background ions and MS as well.  And then again, the last step, some solvents are excellent antibiotics.  Some compounds have an effect on immunosystems and whatever, so sometimes you have some great,  great positive results in your acid, just because you picked the wrong solvent.  So all of these things are very important, but the first thing to consider is the relative  polarity of these solvents.  So again, further down in the polar end, we got water as the most polar solvent, then  we're moving up to methanol or other alcohols for that matter.  That could be butanol, probenol, ethanol, the whole thing, acetonitrile, then we're  moving into the apolar solvent.  So this is where we got ethyl acetate, dichloromethane, and heptane.  So we got the full range of solvents, and we got the full range of polarity in our compounds,  so we just have to mix and match what fits together, and which will fulfill the above  requirements that we have.  So it's very easy.  So we'll start with a little exercise and have a break as well after that.  Oh, after four or again.  Yeah, so we have printed out the exercises here.  So what we want you to do is to try to rank these natural compounds according to their  polarity, both at high pH and low pH.  This is where polarity changed with pH for certain compounds.  So you have to identify the potential functional groups that can be charged at different pHs.  And then we'll go through the exercise afterwards.  How long should we take, Christian?  15 minutes?  Yeah, you can show this one and the next one.  So we take the break.  You need the break now?  Yeah, so you can have the exercise and the break together.  Yeah, in 20 minutes, yeah.  So at two o'clock we'll...  There are many exercises here.  So I can't...  I'm sorry, okay, there's only one with this one.  Yeah.  So 15 minutes, yeah.  15 minutes, yeah.
Chromatographic_Separation__1_a3sDuP0gysU_0_1475.png Chromatographic_Separation__1_a3sDuP0gysU_1476_6755.png Chromatographic_Separation__1_a3sDuP0gysU_6756_7463.png Chromatographic_Separation__1_a3sDuP0gysU_7464_8987.png Chromatographic_Separation__1_a3sDuP0gysU_8988_10271.png Chromatographic_Separation__1_a3sDuP0gysU_10272_11867.png Chromatographic_Separation__1_a3sDuP0gysU_11868_13463.png Chromatographic_Separation__1_a3sDuP0gysU_13464_17087.png Chromatographic_Separation__1_a3sDuP0gysU_17088_17603.png Chromatographic_Separation__1_a3sDuP0gysU_17604_18755.png Chromatographic_Separation__1_a3sDuP0gysU_18756_20087.png Chromatographic_Separation__1_a3sDuP0gysU_20088_20891.png Chromatographic_Separation__1_a3sDuP0gysU_20892_33875.png Chromatographic_Separation__1_a3sDuP0gysU_33876_34079.png Chromatographic_Separation__1_a3sDuP0gysU_34080_34127.png Chromatographic_Separation__1_a3sDuP0gysU_34128_34391.png Chromatographic_Separation__1_a3sDuP0gysU_34392_34439.png Chromatographic_Separation__1_a3sDuP0gysU_34440_35711.png Chromatographic_Separation__1_a3sDuP0gysU_35712_36299.png Chromatographic_Separation__1_a3sDuP0gysU_36300_38315.png Chromatographic_Separation__1_a3sDuP0gysU_38316_38663.png Chromatographic_Separation__1_a3sDuP0gysU_38664_38711.png Chromatographic_Separation__1_a3sDuP0gysU_38712_38927.png Chromatographic_Separation__1_a3sDuP0gysU_38928_39395.png Chromatographic_Separation__1_a3sDuP0gysU_39396_40055.png Chromatographic_Separation__1_a3sDuP0gysU_40056_40271.png Chromatographic_Separation__1_a3sDuP0gysU_40272_43835.png Chromatographic_Separation__1_a3sDuP0gysU_43836_44171.png Chromatographic_Separation__1_a3sDuP0gysU_44172_44735.png Chromatographic_Separation__1_a3sDuP0gysU_44736_45203.png Chromatographic_Separation__1_a3sDuP0gysU_45204_46187.png Chromatographic_Separation__1_a3sDuP0gysU_46188_48131.png Chromatographic_Separation__1_a3sDuP0gysU_48132_49679.png Chromatographic_Separation__1_a3sDuP0gysU_49680_52271.png Chromatographic_Separation__1_a3sDuP0gysU_52272_52907.png Chromatographic_Separation__1_a3sDuP0gysU_52908_53039.png Chromatographic_Separation__1_a3sDuP0gysU_53040_53303.png Chromatographic_Separation__1_a3sDuP0gysU_53304_54275.png Chromatographic_Separation__1_a3sDuP0gysU_54276_54323.png Chromatographic_Separation__1_a3sDuP0gysU_54324_55091.png Chromatographic_Separation__1_a3sDuP0gysU_55092_55295.png Chromatographic_Separation__1_a3sDuP0gysU_55296_56579.png Chromatographic_Separation__1_a3sDuP0gysU_56580_56979.png
Good afternoon everyone. We're starting with a poll. I don't know many of you were traveling maybe last week. So this year we're going to try out some SMS voting and any of you not here last Monday. So it works that way that you lock on to the wireless internet with your smartphone or your computer and unfortunately people with no computers or smartphones cannot vote so they have to have their neighbor help them. Yeah, yeah we won't ask you yet. So but you lock on to this web page up here and then hopefully it will be so. I think you need the three W's and these have to be in capsule. Yeah, so try to lock on and these seven statements should appear on your phones or your computers. So we'll see if it's going to work out today. We have Cecil here from Learning Lab DTU seeing how these new teaching tools are working out and got video recording on today as well. So we're going to try out a lot of different things today. So yeah, try it. Okay, it's working. Take a couple of minutes. And again, there is more than one correct answer so vote as many times as you like. Excellent. Yeah. I'm going to buy a stove so you can get it for yourself. I'm going to buy a stove so you can get it for yourself. I'm going to buy a stove so you can get it for yourself. Excellent. It's working again today. So I'll stop this one. Maybe it's unfair to start with a quiz. But like we talked about yesterday, last Monday, this year we're going to try to use the learning objectives a little bit more actively than has been done the other years. So if you recall, one of the learning objectives from last week was to know the difference between primary and secondary metabolism. And all of these statements have something to do with the common misconceptions or conceptions about primary and secondary metabolism. So five of you voted that secondary metabolites can be found in oral living organisms. And that, in fact, is true. Organisms have different capabilities of producing molecules, but even we as humans produce secondary metabolites, you can say hormones are a sort of secondary metabolites. So that is a true statement. I'm very happy that none of you answered that they are essential for growth, development, and reproduction, because that is what the primary metabolites are involved with. Perhaps as humans they probably are because of the pheromones, but yeah. So yeah, very happy about that. A lot of you agreed that they're made from the intermediates that are coming from the primary metabolism, and that is correct as well. Secondary metabolism can be seen as a second layer of metabolism in that sense. Also very happy, confer a competitive advantage to the producing organism. And in that sense, we talked about last time they're not essential in for growing and living. But they're essential for growing and living better than others and staying alive. Then a few of you voted that they include sugars, fats, and nucleic acids. And this is very interesting because this is not neither true or wrong, because we have something of a gray area, in particular with the sugars and fatty acids, not fats, but fatty acids. And that is very much in the gray area between primary and secondary metabolism. Are produced under all growth conditions? None of you voted for that? Excellent, that is true. Secondary metabolism is very much dependent on the growth conditions, and are the same in all members of the same species? None of you thought that either? That is also correct because it's actually a way that we can differentiate different species. Excellent, well done. You fulfilled one of the first learning objectives of this course. So today it's going to be chemistry day. You're all excited about that. So we're going to start talking about chromatographic separation of natural products, secondary metabolism. And one of some of the things that we're going to teach you today is to talk about liquid chromatography. Christian gave a quick overview last time, but today we'll go more into the theory behind it. And Christian will explain to you how the different components of an HPLC works. Also we'll talk a lot more about functional group chemistry. Last time you had a little recap on functional groups because natural products are particularly rich in functional groups, and this is exactly what we exploit in chromatography. And then we'll talk in particularly about silica as a stationary phase and reverse phase separation because this is one of the things that you're really going to learn a lot about in this course. I'm going to ask you about for your exams and you're going to use it for your exercises. So hopefully by the end of today you'll be able to outline the main mechanisms that we use to separate compounds. You'll be able to highlight the different types of chromatography. Again here in particular reverse phase separations, but also something called helic and normal phase separations and explain when are we going to use them, for what type of compounds are they particularly useful. And then you're going to do a lot of prediction. Just from looking at structures and molecules, how would you predict and expect them to behave in a chromatographic system? So we'll have four parts today and it's going to be a little bit of mix and match. So we start out by talking about functional group chemistry and then you're actually going to have some group exercises. You can move around, move to the next room because we realized that last time this is a very small room with very little oxygen in it. So move to the next room. It has oxygen and it will have coffee and cake as well. So yeah and then Christian will take over also have some lectures and some exercises in between. So hopefully it will break up the day and not be too tiresome. So some of the things that we talked about last time is what we have a plate of microorganisms that are capable of producing a lot of different molecules. How, which steps would we go through in order to figure out what they're capable of doing? And that goes for both known compounds that could be toxins or it could be completely new molecules that we want to use for new drugs. And even though we have a slightly different approach depending on whether or not we know what we're looking for or we have absolutely no clue what we're looking for, we go through some of the same steps in the analysis. And you came up with some great suggestions. And the essence of those steps that you're all listed were extraction, separation, detection, identification. And today we're going to talk in particular about that part here in the middle, separation but also a little bit about detection. And then I'm very fond of that statement up in the corner here saying isolation, purification, separation, identification and celebration because that is exactly what this is about. When you actually manage to go through these steps, we have a party. So when it comes to separating compounds, what chemical characteristics can we actually exploit? Do you have any ideas for that? If you have these three different compounds, how are they different? How can you separate them based on their differences? No? We have three major chemical or physical characteristics that we use. First of all, we can use their polarity. How soluble are they in water? So that is a major characteristic. Any suggestions other than that? Yeah? Exactly. So some compounds can differ quite a lot in size. And here again, of course, we see the two-dimensional structure. Size will play an even bigger factor when we've got the three-dimensional structure because some compounds can fold in and make globular shapes and whatever. So size is a really good way of separating compounds as well. Any other than these two? If you got functional groups like this, some of them are capable of carrying a charge. So that is the last major thing that we can separate compounds based on. If we're talking about compounds that we know that have a specific biological function, we can also use affinity. So we can separate compounds based on whether or not they will bind to a specific protein or receptor. But that is not something that goes for all molecules. That is not something that we can use as a general separation principle. So in this course, we'll go through the three top ones here. And the reason why we want to know all the mechanisms that we can use is that sometimes compounds do not differ a lot when it's just looking at one characteristic. So for example, you can have compound A and compound B, which are very similar in their polarity. So no matter how much we optimize the different solvents and how we will never be able to separate these two. However, so we introduce second dimension, the size, and the third dimension, the charge. And in particular, the charge, whether or not something have a positive or a negative charge, that is really something quite distinctive of a compound. And this is what we call orthogonal separation. Because in theory, these characteristics does not have anything to do with each other. That is not entirely true. Because the polarity will also depend on the size of the compound. Usually a very big molecule, if all functional groups are the same, that is more A polar than a very small molecule. And also the charge can change the polarity of a compound. But I'll return to that. So when we're talking about natural products, exploiting functionalities is really important. We want to make the best of what we know about these compounds. We don't know a lot, but we know they have a lot of functional groups. And in fact, if you make statistics about all microbial metabolites that are known today, you can see this distribution. You can see that if you look at compounds from filamentous fungi and bacteria, and you should see the total number as well, that also includes algae and other types of microorganisms, you can see about half of them, they have an acidic functionality. That could be an enol or phenol that is capable of exchanging a proton or simply a carboxylic acid. And the carboxylic acids are about half of the acidic functionalities that are present. Then amines, they can be positively charged, they can act as basis, not if they're close up in a ring or in an amide, but otherwise they can carry a charge. And you can see here about 28% of all bacterial metabolites, they have a cationic charge. So this is really, if you want to sort out and separate out compounds, you can remove almost a third of these compounds just by looking at a positive charge. So these are the functional groups that we talked about last time. And we try to exploit these functionalities when we look at chromatography. So Christians showed you this slide last time as well. This is a picture of some of the beads that we have in the stationary phase of chromatography. So this is what our compounds will bind to when we try to separate them out. So by packing a column with a lot of these different particles or beads, you can tell that if you change the selectivity, if you change the affinity towards specific functional groups, you can actually change how fast the compound can move through a column. So this is how a particle looks on the inside. So when we're talking about reverse phase chromatography, but also normal phase and hydrophilic interaction chromatography that we'll discuss today, the base of the particle is silica. So we have a lot of different cross links within the particle. And then we can have different changes to that silica particle. So we can introduce, here we've got a long C18 chain. And this is the basis of most reverse phase separation, that is C18. So it's a long aliphatic chain, which is very apolar. So that will attract very apolar molecules. So compounds with very polar functionalities, hydroxyl groups, amines, whatever, they will usually be repelled by this C18 chain. But in principle, it can be anything that you can change here, the R group. It can be hexyl, phenyl, amino groups, cyanogrups, diol groups. You can put everything on that will make your compounds stick to it. So you can change your selectivity towards specific compounds if you change the chemistry of the particle. So how many of you have heard about chromatography before, except for last time? Now you had a few. So I'll show you the very cartoonish explanation for the rest of you. The principle behind chromatography here again is that you have a stationary face. This should reflect the particles that you saw in the other picture as well. You got the round beads, which are functionalized with different types of R groups. So here is C18. And Christian mentioned the POP-CROLE model of chromatography last time. These are the bouncers. So these are the ones that decide who gets in and who gets out of the bars and how long it's going to take. So you have a mixture of analytes up here, and they will move through the column with a mobile face. So everything in chromatography is about are the compounds most fond of the stationary face or the mobile face? And the mobile face is moving at all times. So you've got a flow coming through here. So you'll have a constant flow of mobile face, which can attract your analyte molecules, or they can be absorbed into the stationary face. And then you can see that depending on the functionalities of your analytes, they will move faster, slow through the column. They will bind weakly or strongly to the stationary face. So maybe you're already guessed by now that the stationary face, the particles that make up the stationary face, they are really the essential part of chromatography. This is where we can make all the difference. This is where we can buy different columns and obtain different kinds of separation. So here we have two different kinds of particles. And you can see that they differ a little bit in their structure. And actually, there's so much different from brand to brand that you can get selectivity just from switching to another vendor than changing the stationary face. Even though they're both C18. So even though you've got the same R group sitting on there, the way that the particle is constructed will actually sometimes matter even more. So when you will read papers about how compounds are separated and you say that they, you can read that they use the particular brand, Phenomenics, something column, you can't just go out and buy a different brand and expect exactly the same separation. So in fact, when you got the picture before, you got the compounds passing through the column, they will not just interact with the surface of the particle. In fact, you'll see the compounds moving into the particle. They'll be absorbed into the particle. And we got this porous layer, older columns. They have a particle that is completely porous, which means. Like sponge, you know, in the bathroom. Yeah. So, so in fact, that's the compounds can pass all the way through the particle. And that of course takes time, which makes sometimes very unpredictable how compounds will behave with a certain particle. But today, most modern particles, they have like a core, a heart core shell, and then they have a porous shell on the outside. And the particle size is then you got, in this case, you got a 0.35 micron porous shell. Here you got 0.23 porous shell. So that the compounds from the analyte will only move through the porous layer, which means that it will go faster and you can get better separation. So again, if you buy a new column with C18 that is constructed this way, you will have a much different type of selectivity, a much different type of chromatography compared to an old C18 type particle. So the chromatographic mechanisms that will go through in this course, they're all mainly based on separation based on polarity. And we have the scale compounds going from polar to a polar. And how polar or a polar a compound is will very much depend on the functional groups, like I said. So right here in the very polar end of the scale, you got salts. So if you got compounds with a permanent charge and you got a counter ion sitting there, these will be very, very polar. You got different types of acids, carboxylic acids are the most polar ones, but then you got alcohol groups, ketones, ethers, halogens, aromatics, aliphatic compounds, and then fluorinated compounds all the way out to the end. These are usually the ones that you'll encounter in this course. And maybe some of you remember from last time, we had a pole with a natural compound and a synthetic derivative. And the synthetic derivative had floor stuck onto the structure. So this was a strategy for the pharma company to increase the polarity of that compound. So by introducing different functional groups, you can change the polarity of a compound. The scale that we use to assess how polar or non-polar compounds are is called LOC D. And it's a logarithmic value. So you see the distribution of compounds between water and octanol. So of course, the octanol is in the top one. So the lower the number, the more polar, the more of the compound will be in the water phase. So that means that you can actually have negative values. So if all of the compound is, will be in the water phase, you'll have a negative value as well. Here you can see the effect on different functional groups on the LOC D value. And this is just an assessment. Modules will not always behave as linear as you would expect them to do. Some of them do. But it's a good, gives you a good feeling of how the different functional groups will affect the LOC D value. So if you got a hydrogen and you exchange that with the hydroxyl groups, you'll have a change of minus 0.67. So on the other hand, if you introduce an aromatic group, you'll see an increase of 2.13. So you can actually predict sometimes how the polarity will be of different natural compounds. So here you got canamison, an antibiotic made of free rings of sugar, very high of the sire to be in the water phase. So it will have a negative value of 12.6. Then we got another antibiotic, TDA, trebotithiac acid, also very polar compound. Just for comparison, we'll put ethanol in there maybe. That is a better indication on the scales that you're used to. Then we got a mycotoxin here, which is medium polar. The condom, the spice, the chili, the compound that will give you that burning sensation. This is the quiet A-polar compound. This is one of the explanations why it helps to drink milk. A lot of fatty things, drink a bowl of fat and then you won't feel the spiciness. Then also a toxin, also very A-polar, and then you got bicaratine, which is all the way in the other end of the scale, very aliphatic molecule. So it's very, very A-polar. But I mentioned in the beginning that these free principles that we got to separate compounds, they're not completely orthogonal. That is because that pH can affect the polarity of the compound. That is because compounds with charges, they have pKa values. They can change the charge depending on what the pH they're in. You had TDA before, which was a very polar molecule. In fact, if you change pH, it has a pKa value of 3.7. So at high pH above 3.7, it will lose that proton sitting here in the carboxylic acid. This will give it a negative charge, which will make it very soluble in water. On the other hand, if you decrease pH, you give it a lot of proton H+, you make sure that the carboxylic acid is protonated at all times, which will make it more soluble in organic solvents. It will make it more A-polar. Make sense? Yep. Again, here we've got Rogfortzen. It got an amine sitting there. It can carry a positive charge. It will do that at low pH, so if you've got a lot of H+, this will be N+, which makes it very soluble in water. Again, if you increase pH, it will be deprotonated and more A-polar. So if you want to extract this from water, we'll easily acetate or hexane or something like that, and you have to be at high pH. Here, we have to go low. Yeah. Exactly. By knowing this about your compounds, this is a very selective factor that you can use. Then, of course, you've got ethanol. It does not have a charge at any pH, so you have a constant lockdown value. It will not change. It will actually up here, but it can't calculate it. It will start to make a negative charge, but at the extreme, it is in concentrated sodium hydroxide. Yeah. But at natural pH values, it will never be charged. We're talking about C-lock B value, which is a pH-adjusted value. One thing, this is a calculation tool we have. This is not experimental data, but we have computer programs. We actually have a campus license, so this we can just calculate. It may not be totally true, but it's going to be pretty close to... Yeah. To what's right. Yeah. So, using something like the table that we showed before, that you will see an effect of something-something. It's based on some experiments, but some compounds are better predicted than others. Rural foam, acids will be more A-polar at low pH, and bases will be more A-polar at high pH. You got the scale again from polar to A-polar, and why are we interested at all in knowing why our compounds... If our compounds are polar or A-polar, that is because this defines the type of chromatography that we're going to use. So you'll see the big hat up here, that is reverse face, and that covers actually 80% of the polarity range, something like that. It will cover most natural products. So this is why you have to spend so much time on learning about reverse face chromatography, because this is actually the most versatile and the most commonly used method. But you're also going to learn about normal face separation, which is in the A-polar end of the scale, and you're going to learn about hydrophilic interaction chromatography, which is other end of the scale. Using the extremely polar compounds like the sugars, canamysin that I showed you before. And everything inside the cell, if you recall you by chemistry, you would see that actually all the things in the TCA cycle, all the phosphorylated compounds, I mean, they're down here, and if you inject them on a reverse face column, just going to pass directly through. So if you're ever going to look at primary metabolites, that is definitely in this end of the scale. Then, not today, but maybe later. Haven't decided yet. You're going to learn about other types of chromatography, ion pair and ion exchange, but very briefly. But again, it's important to know which type of column would you pick out from the shelf if you want to analyze a particular type of compound. Yeah, so special emphasis in reverse face, and then something about normal phase in hydrophilic interaction chromatography, but most importantly, that you will be able to know when to expect reverse face to fail, and when do we have to go dig somewhere else on the shelf for something other than that. So also talking about polarity and chromatography, this is very big part of that is the mobile phase, the solvents. But we use solvents for a lot of different things. We use the solvents for extraction. We use them as part of the HPLC system. We flush solvents through all of detectives, and we actually use solvents when we want to run bioassays, for example. And solvents are very important, and even though that you're not chemists, it's very important to know what type of compound that you're dealing with and what solvents that you can use. Because one of, in our many collaborations, one of the biggest questions that will arise every time, we as chemists have produced a compound that we want to test in an antibacterial assay, and then the people running the assay, they will come and ask, what should we dissolve it in? And we will like you, for people that most likely will be the one running the assays, to know what solvent to put your things in. And sometimes things are not always compatible, because we have certain requirements for the solvents that we're going to use to dissolve or sample in. So maybe we have a compound that is not soluble in water at all, so we need to use a more apolar solvent. But when we start to analyze it with the HPLC, we also want to make sure that it's actually mixable with the solvents that we have in our system, that it's not making two-phase systems, that you got your sample floating on top of your mobile phase running through, then you won't have any of that fantastic functional group chemistry, because it will be isolated in a little bubble. Then we have something called illotropic strength, which is the power of a solvent to take the molecules with them in the mobile phase and take them through the column. We also have to consider viscosity, because that will influence the pressure of a chromatographic system. Then when we are passing them through the detector, we also have to know whether or not the solvent itself will affect the detection. So if we got a solvent that has great UV absorption, it doesn't matter how good our natural compounds are at absorbing light, because they won't see them at all. We have a huge background peak. That's why we don't use acetone, because that takes all the light up to 350 nanometers, and that's where we see most of the goodies. So this is extremely important, but we also have some solvents that will cause a lot of different annoying background ions and MS as well. And then again, the last step, some solvents are excellent antibiotics. Some compounds have an effect on immunosystems and whatever, so sometimes you have some great, great positive results in your acid, just because you picked the wrong solvent. So all of these things are very important, but the first thing to consider is the relative polarity of these solvents. So again, further down in the polar end, we got water as the most polar solvent, then we're moving up to methanol or other alcohols for that matter. That could be butanol, probenol, ethanol, the whole thing, acetonitrile, then we're moving into the apolar solvent. So this is where we got ethyl acetate, dichloromethane, and heptane. So we got the full range of solvents, and we got the full range of polarity in our compounds, so we just have to mix and match what fits together, and which will fulfill the above requirements that we have. So it's very easy. So we'll start with a little exercise and have a break as well after that. Oh, after four or again. Yeah, so we have printed out the exercises here. So what we want you to do is to try to rank these natural compounds according to their polarity, both at high pH and low pH. This is where polarity changed with pH for certain compounds. So you have to identify the potential functional groups that can be charged at different pHs. And then we'll go through the exercise afterwards. How long should we take, Christian? 15 minutes? Yeah, you can show this one and the next one. So we take the break. You need the break now? Yeah, so you can have the exercise and the break together. Yeah, in 20 minutes, yeah. So at two o'clock we'll... There are many exercises here. So I can't... I'm sorry, okay, there's only one with this one. Yeah. So 15 minutes, yeah. 15 minutes, yeah.
isolation, purification, separation,