So, please remember that it has to be conjugated double bond for the UV detector award, so it's every second bond that needs to be a double bond, and it doesn't necessarily need to be a

CC, it could be a CO bond. And normally the more conjugations you get, the more peaks you get. But the best key is still the further out in wavelength, the higher wave absorption, and a higher

nanometer range, you will actually have to absorb. So they're going to move and move, and about from about 450 nanometers, 400, 450, that's the visible range. And for the fluorescence,

say that's only about a third, and you use very specific wavelength to get the electrons up, and they're going to send out a very specific wavelength the other way. That's what gives you the specificity. So the downside of fluorescence is that

first look at them, if you have any conjugated double bonds, and if not then it's not going to work, but you still have to optimize it, so you have to find an optimal excitation wavelength and an optimal emission wavelength. So it can take a long time. On this slide, this is the analyte. This is the region.

So when you look at these two molecules, I think the easy thing you should remember to the exam is that the more conjugations you have, the further out in the wavelength spectrum you have. So this one is of course this one,

and then this one must be this one. Then I agree, then which one is coming first, and if you calculate a log-D, you will see this one is the first, and this is actually a true chromatogram

with these peaks in. It's not something I made up. It's a true analytical separation, and the reason is that size also matters. For such a last molecule, if just one piece of the molecule sticks to the stationary phase, it doesn't move.

But again, if I start asking in which order stuffs like this come at the exam, don't worry, then it's because we are up in the good end.

So this one is X, and this one is Y. Sorry. So if I say that there's probably only one we can detect of these,

which one? Here we have our conjugated double bonds. This one you can absolutely not see, and these two especially this one. If you inject a very high amount and have absolutely just pure solvents, you're going to see a very small peak, but this is a bacterial signaling molecule, and

you cannot detect them in any biological concentration here. So these are, you can just see, but you need really, really a lot to see it, so it's not feasible detection. So only this one is the solution.

Then I'm sorry, this is an old assignment. We actually changed to make it simpler. Well, we showed a number of structures. So the idea is, this is first coming out of the HPLC, and then it goes into the UV. This is the signal we get here.

So this is a plot from the software, and we get the data in. And then the evaporative light scattering, which you can see more. You can actually see the strawberry extract. So a peak must first appear here, and then here.

On the other hand, you know, there could be compounds only seen here, and not seen here, and the opposite. You know, each detector sees various things. So it has to be, and also these are liquids. So liquid is always flowing, first of name, slow cell, and the next.

So it has to be a constant time delay, and I think this is a time delay. So you see this peak is not detected down here. This peak must come from something which is not containing any double bonds. But you will always have the same time delay

during a run. So of course, to characterize this, we of course don't take a mixture, but a compound, you know, is UV absorbing, and you also think will be seen by evaporative light scattering. And then you characterize that, and that will be a constant time,

because that's only going to be a little bit of peak tubing from one detector to another, and that will have a very low volume. So you should have a very low time delay. In this case, about 0.1 second.

So for any assignment, always remember to start on the easy stuff, and then get, and then let's get things made more difficult.

So this one looks like it has a lot of conjugated double bonds, so which one will this one be? This is number three. And what, yeah, what, which one could else be easy if we take this one, for instance, so which

peak will that one correspond to? Then, so that's the, that's the two easy ones. Then there's one with absolutely no conjugations, so which one can this one be?

Right, and then things get difficult, because what's the difference between these two?

So we call this part of the molecule the chromophore. This is a part of the molecule we can see with the UV detector, and these are actually identical. This is what they're just drawn slightly different. So the solution is we cannot, based on the UV,

differentiate one and four. So this can be one and four, and this can be one and four. It's very important as an engineer that you don't over interpret your data. Sometimes the result is, I don't know, I cannot differentiate these things. That can be a very important statement.

And then I guess there's one left. That's this one, and it doesn't have many, and this is, these are two spectra from our database. So I guess even though these are not conjugated, something happens with the oxygen here, and it is actually seen.

So which ones of these two will not be worth the time to check for fluorescence?

Yes, they will clearly not make any kind of absorption.

These two, well, that needs to be checked. This one is actually very well detected with fluorescence. Usually planar molecules or lots of conjugations, you know, you may find good fluorescence.

Okay, so give it a few minutes. Let's discuss a little bit with your one next to you.

Have you ever heard of this used? Do we use this anywhere in the world? I'm sure you have heard about things in the press that will probably be made.

Any ideas of where, is this ever used, or is you just going to learn something useless? Any ideas? So you're just going to learn something. We can just as well go home now.

Yeah, it's a very good thing if you know what you're looking for. Anybody have been on the engineer's homepage, the Danish Engineering Association?

Lately there's a debate going on on fluorinated compounds and in all the paper products we have. It turns out there's no legislation for whatever put our our food products in. If it's paper, if it's plastic, there's gasoline. So things you have to apply to and not be toxic and test and test. If it's paper, no problem.

So this turns out that these fluorinated, so they have some long chains, well they got flora.

Sometimes they have various stuff here that is a polar group or something. We also know this, some Teflon, these things. And you apply this, you can actually add this to paper and bind it pretty strongly. And this is why you can have some paper where you put oil on and nothing happens.

And the bad thing about this shit is that it goes out to the food and it's probably not very well, it's probably modulating our some of the intertwined systems, some of the things to do with the various, they can pretty much work as hormones also.

So it could be very, could be the biggest scandal we have had in the food sector in the last, I don't know, 10 years. This is why you shouldn't eat popcorn from the microwave. I think this is going to be a really, really big problem and the industry is doing everything to cover it up.

This is mass spec detection. Often they have assets, so we're working in negative mode, we'll come back to that. This is a place, yeah? Could there be other places where we use this? We think this is used in sport.

Or have stuff associated to sports? Yes, so this is the, so the Clint Bottero found in, I don't know,

yeah, this famous one at the Danish team. He was, he was, he was nailed by mass spec detection. Mass spec detection is actually a very, you can do a very clear identification, you know, you can do something that holds in court. So most stuff

that, if you're talking about unwanted substances, if you need to document, you will need to document with mass spec detection. This is very, very specific detection and we'll come back to there, many other places.

Yeah, well, as we said here, is is Clint Bottero role in the blood or in the urine? Is the fluorinated compounds, are they present in the food matrix? You can have a reference standard, you can, you can also check the methods.

So this, if you want to quantitate, this can be a lot of work, because you need to have the reference standard, you need to have, you know, I have a certain amount, I need to be able to add to the sample and I need to be able to show that I can find it again, so I, that way I validate my method, I show that it works.

So you know, the biggest difficulties in analytical chemistry is to say, it is not there, but if it was there, I would have found it. Usually no problem to show something is there, but then

where you see them used a lot, if you go out to the big pharmaceutical companies, you will see mass spectra takes us all over. For instance, you will, if you have a nice drug, you would need to approve what happens if you

give it light or heat or you need to show the stability of the compound and you need to show what is it degraded to. So you need to identify all the degradation products and you will actually also, after that, you will have your synthetic chemists to produce more of them and you will also have to prove that they're non-toxic.

You will also, because you do a product where you do a fermentation or you do a chemical synthesis, you would need to identify all you need to identify all contaminants or other peaks in the product.

This is actually how Lundbeck, they use a lot of mass spec and that is mass spec in court. I don't drag it there, literally. But you know they have this patent on Ciprolegs, I think it's called, this antidepressant. There is some companies who claim they have, they don't have a patent on the compound anymore,

but they have a patent on how to produce it. And they know when we produce this, certain impurities will occur and they will always occur when we do the synthesis this way and we have a patent, so they simply get the competitor's product and analyze them and find these impurities.

That's why they still make a lot of money because they have good chemists that can take this and hold it in court. Then what happens if we put a drug into an animal? Is it just going out in urine? Well, they're

changed. Go into the liver, we have all the cytochrome enzymes. They're going to oxidize stuff. They can, if it's not very water soluble, it could be conjugated to various stuff. That's a chemical change.

So again, if you have your drug, you use your mass spec to show how are they changed. You will also need to show that they actually go into the animal. That's the biggest. That's how you kill most drugs. 95%, when I've worked at neurosurge, 95% of the drugs we had in, they did not go into the animal. You fed them,

you know, in the stomach and we have to prove if they went into the blood and if they went into the brain or not. That's all mass spec. And of course if you're working with pesticides or stuff, it could be that, oh, you say we cannot find this pesticide in the environment.

Yeah, yeah, but what about degradation product? So if you come with a new pesticide, you have to show how it's degraded and you will also have to have methods for not just detecting the parent compound, but also all the degradation product. This is all mass spectrometry. But then of course you can have something totally unknown.

It could also be up here working with pharmaceuticals. You certainly have a new strange peak in your chromatogram and you will have to do an investigation. And if it's a batch of the final product, you have to hold it until you know if it's a toxic compound or not and

they spend a lot of time on that. But the mass spectrometer compared to the UV detector, the mass spectrometer can tell us the mass of the compound. If you have certain types of mass spectrometers, it will tell us the elementary composition. This means we could say this is a Z20H32N2O5.

This could be a great help for you to know where the hell does this this impurity come from or where is the likely source. So the mass spec can tell you all this.

So a very important thing about a mass spectrometer is it can only measure ions. Means that most of our molecule things we are working with are neutral molecules, so we have to ionize them. If we don't ionize them, we don't see them.

So the first step going to the mass spectrometer is simply the ionization chamber. We can analyze and we can add charges in various ways. So you will work a lot with electrosprays. So you will probably also mainly work with positive electrospray and we'll see if we can add a positive charge.

This could be a proton, but it could also be a sodium or ammonium or pysidium. So here we have a neutral molecule and we add a charge. Then the whole trick is then putting a charge on and getting it into the instrument, which is a high vacuum system.

We could also remove a negative charge. So we have a neutral molecule and we take off an electron. We have a positively charged ion. This is a very specific ionization mode that is used a lot in what we call electron impact mass spectrometry.

That is very often what you see if you have gas chromatography combined. This is where you see the big library searches. This is probably what you see in these CSI series on how they found this. Because this will make a very, very reproducible ionization and a very good fragmentation of your molecule.

And so you can make big libraries and you can make it on different manufacturer's instrument and get the same mass spectrum of the same compound. This is not the case up here, unfortunately. And I can also give you a guarantee. If you could get this technique to work with HPLC, you will win a Nobel Prize.

So that's the challenge. Because this is so beneficial. This is why all labs also have GCMS. Because if you can get it through the gas chromatography, many molecules won't. But many will get them through.

You can in many cases at least see if it's been seen before. And you can buy big libraries of up to 250,000 compounds. We could also, again this is usually with electrospray. Now it's negative electrospray. We can remove a proton and then of course get an M-8 ion.

And we can also, most instrument can work in negative mode. They have to work in one mode. But some can switch all the time. So you can do negative for 100 milliseconds. You go back 100 milliseconds to positive, negative, positive, negative, positive.

And if you can do that fast enough compared to your chromatography peaks, it can be very, very beneficial. Many of the very special instrument can measure the mass very accurately. They cannot do this. Some neutral molecules we can actually also just add a negative charge.

It could be formate, missing NH here. But formate or acetate or chloride, put it on neutral molecule and then you can see in a negative mode. Another important statement here, we never measure the mass. We actually always measure the mass to charge.

As long as we're down on very small molecules, we mainly have singly charged. This technique will always generate singly charged ion. Electrospray can actually produce multicharged ions. So if you work with proteins, you will apply many, many charges.

That means that if you have an ion that has the mass with all the charges of 10,000 and you have 10 charges on it, you're going to see it as a mass to charge of 1,000. Luckily, there are tricks where we can look at the ions,

the ion clusters, the mass spectrum, mass spectra and see how many charges do I have on my molecule. When it's over, always M over Z. And for some strange reason, if you say that I have an ion of 200,

you say M Z 200. So we measured this ion. We don't put the M Z after. Some historic thing. This is just how it is.

So now we're going to some of the little bit tricky stuff. I have to say isotopes. And now we are mainly talking stable isotopes. So this is how it looks out in nature.

So you remember hydrogen. And I know there's deuterium and tritium out there, but they are so rare we don't look at them. So the mass of a proton is one or hydrogen, or actually it's 0.007.

And if you have an instrument that can measure the mass very, very accurately, as some instruments can, we can use the decimals for something. Whereas other instruments that cannot measure this accurately, you can't use the decimals for anything. But you see, if I have 12 hydrogens,

the mass will not be the same as one carbon. Also very important, monoisotopic. We always use this term because you're used to using the molar mass.

And that is the average. Because whenever you have 100 carbons, you're going to have 98.9 carbon-12s. You're going to have 1.1 carbon-13. And this is why you know the molar mass of carbon

to be 12.012 or something. It's because when you get a lot of, when you get some compounds, some sugar, you will know exactly the distribution, but you will have both types of carbons in that sugar,

unless you bought some that is isotope enriched. You can also see with chlorine that you have chlorine-35 and 37. And because 75% is chlorine-35 and only 25% is chlorine-37,

that is why you know the molar mass of chlorine to be 35.5. Because that is a weighted average. Sulfur is also found. What is actually very interesting with chlorine,

sodium and bromine, is that you see the mass difference is two. It's not one. Up here, the mass difference is one. Here, the mass difference is two. Here, the mass difference is two. Here's a two. And that are very important detail that we can use so long.

As I said, in some of the instrument can measure our mass very accurately. We call that accurate mass. The instruments, they can only say this ion is 200. We call those nominal mass. But you can also see that if you can measure this very accurately,

you will be able to differentiate these two. We'll come back to those next time. You can also come back to this also next time. We can also express if this is a mass defect.

So to see what is the difference, like oxygen actually dragged down the decimals, fluoride dragged it down much more. So the sulfur and bromine was hydrogens go up.

So if you look at lipids, they're mainly hydrogen and carbon. Up at about 500 or so, you're going to have a decimal at about 0.5. Whereas if you had something with very few hydrogens in and a lot of chlorines,

the decimals could be 0 or it could even be that it was 499.9. So we can actually, in some cases, we can use this. Let's do some drawings.

Yeah, I was supposed to use the, you get some white ones because I was supposed to use the electronic board, but this one teases me.

So let's look a little bit into some ethanol. So this is ethanol, and we got some different what we call isotropomers.

I can tell you that we do actually have four of them. So which one will be the one we have the most of?

If we look into carbon 12s and carbon 13s, if we have one with 12 here and a carbon 12 here, this is probably going to be the most common one. So this one, there will actually be 98.9 to the second of these two.

And then this one, we could also have one where this is a 12 and this is a 13. We can have one where this is a 13 and this is a 12.

And we can have one where this is 13 and this is 13. And of course, the change here is 1.1 percent to the second. You're not going to see much of them. And these two, this is going to be 98.9 multiplied by 1.1, and the same here.

And the interesting stuff here is this one has the same mass.

So if we want to create an electrospray spectrum of these, oh sorry, an electron impact where we just take off an electron, then let's see, what do we have here? So it's 24 plus 5, 29, 17, that's 46.

So what's interesting on the mass spectrum is here, now we have M over Z. And we could now take a lot of, lot of, lot of molecules of ethanol

and charge them, let's say we steal an electron. Then we agree we should have one that has the mass of 46, because these are, these up here, this is about 100 percent,

this is about 1 percent, and this one is also 1 percent. So, oh, so now we're going to have 46, we're going to have one here with a mass of 47, and this one is going to be about 2 percent.

And we're going to have a tiny one, it's not going to be big, because it's going to be 0.1 percent of a percent, so that's 0.01 percent.

So we're going to have a very, very tiny ion here, we can hardly see, we probably won't see unless we really load a lot into our instrument. This is called an isotope pattern.

And again, if you take the weighted average of this, you're going to get the molar mass. So it's very important that any molecule that contains a lot of carbons,

they will have an isotope pattern. This is a long way in memory, I assume. Let's take something more complicated.

We take, now we're starting to get combinations.

So we could have one, well this is still a 12, and a 12, and a 35.

Now this one will actually be, if we only have these two, we will have a mass spectrum, and then we're going to have,

can you recall how much did we have of this one? 75 percent, and 76, and about 20.

Oh sorry, yeah, and 24. So anyway, we're going to have, we have to have a mass difference up here. So now we have a spacing of two here, and we're going to have some mass,

and this one will be two bigger, and this one will be approximately one third of this one. But there will be other combinations, right? Because we must have some that has a 13 here, and a 12 here, and a 35.

And are there some that have the same mass? Yes, we have a 12C, and a 13, and a 35 here.

So these will still be about two percent, so they're actually going to add here two percent.

So we're still going to have a mass difference here of two, but here we of course it's going to have one, and one. And then of course we also have some, where this is a 13, 12,

and we're going to have one that is 12, 37, 13, 13, and we're going to have one that is

13C, 13C, CL, 35.

We're going to have these two with one carbon and 35. They're going to come here, and this one there and there will also come here. And then I'm actually missing one combination. The very, very tricky combination.

Perfect.

That's an interesting one, because 13, 13. This one we actually do have to add here, but it's not going to add more, much.

But it will add up. We could also take some with more carbons. This would kill you. So here's actually a simulated spectrum of a slightly larger molecule.

I can tell you that luckily there are lots of software packages that can calculate this for you. And here's just the drawing. This is actually chrysofovin in the lecture spray. This is why.

So here you see, here we have that. That's one chlorine in chrysofovin. So you have the one with only carbon 12s. You have 12 carbons here. So this one is, no, sorry, there's actually more in that one.

OK. I didn't write the elementary composition. But anyway, we have one chlorine in here, and actually you can actually more or less take this one, and this is like 18, 19 percent. And if you divide it by 1.1 percent per carbon, you can say there's probably at least 17 to 18 carbons in this molecule.

So from the isotope pattern, we can actually say something. We can say if there's bromine or chlorine or sulfur in, and we can also estimate the number of carbons in. That's without all of the symbols. We can add them also. We have a more advanced instrument. We can say a lot of an unknown peak we identified.

Also, your mass spectrometer should always give you this isotope pattern. If you have a mass spectrometer and the isotope pattern is not fitting, your instrument is not working well, and it will not be able to quantitate or do anything.

So an example. You have a molecule. We'll come back to why this should actually read sodium. We have our protonated molecular ion. We can see a third here.

So by this, about a third, we can say we have one chlorine in. We don't have two. If you have two in, it looks differently, because then you again have more combinations up here,

and it's going to be a nightmare. Any mass spec book will have usually have the printed isotope patterns of one, two, three, four chlorines, one bromine, one chlorine, one bromine, and so on, all the combinations, unless there are softwares to calculate this. I think the important thing is to see that we could call this an A plus two element.

But you see here, you have this sudden jump of two. This is not normal. So the other thing is you can go in here,

and I think the rule is now or you can't see the scale here, but I've taken it to about 22 percent. And if you divide that by 1.1 percent per carbon, you get about 20.

So again, if you're working with something, you can estimate the number of carbons very fast. And I also have to say that this model, this simple one only works up to about 40 percent of 40 carbons. Because actually, the way you calculate this, you assume that the carbon 12s are 100 percent.

They're not. They're 89.8. And simply, if you come up to molecules with 100 carbons in, it's also unlikely that you randomly can draw 100 carbons without drawing one carbon 13. That is also going to be slightly unlikely. So I think it's about at about 80 and 5 and 90 carbons.

Then this one will come up there, but you're also going to have a much higher mass because 80 or 90 carbons multiplied by 12 is also about 1000. Then you have to add some hydrogens or whatever.

So again, you can, this was the one we saw before. If you're in doubt and you will have the software or you can have the software that also, when you look at your assignments, you will also have a software package on it that can calculate the isotope pattern. And so you can also go in and test,

you know, is the theoretical one here? Does that fit with my mesh spectrum? And on the instrument, you will be working the data that will be created for you. It's also making a very accurate isotope pattern. And that can be very beneficial in many other cases.

If you're working with microorganisms and you want to see how it changes things, how it metabolizes things, is it, for instance, is it taking up, this is a compound containing an amino acid. If you want to see if it can take up, which amino acids can it take up and use for the biosynthesis of this?

You can buy carbon 13 labeled amino acids. You can feed that. And if you do that and it can actually take them up, you're going to see change here. Of course, if you feed the organism with only the amino acid, then it also will go into the central carbon 13,

the carbon metabolism, it may be more difficult. But if you feed it with a lot of normal sugar and then you have some labeled amino acids, then you can see if this happens. Because then if you put in, let's say, an allylane with six carbons labeled, then you should see a peak coming six masses higher.

So in biological sciences, we can actually use the mass spectrometer a lot in elucidating biosynthesis. And the nice thing about carbon 13 is that it's not radioactive. It's the carbon 14 that the archaeologists are using. That is radioactive. There you need to be in an isotope lab.

Carbon 13 is just as stable as carbon 12. And a lot in the future, you will see increasing amount of biological problems that we can solve with using stable isotopes. You also see this in protein chemistry

where you do different experiments and then on some of the experiments, you use labeled amino acids. And then you can look at all the mass spectra and compare them and see which ones are changing. So again, a very, yeah.

Let's take a break. Until five minutes to, yeah, in 10 minutes time.