All want to go home I Assume you're not gonna stay here for the rest of your life. So any idea of what could happen here? Why don't get such a broad peak When we inject it when there's a lot of water in the HPLC where this compound should be retained

well, so what happens here is that This compounds It's in the citronitrile water it comes into this small

piece of tubing The water and it precipitates out This is actually It's a lot of the blue bacteria that that makes this they kind of makes it as an armor outside their cell membrane and

If you do that, you don't want a compound that is very water-soluble No, it's too expensive to produce So this is actually kind of a medium polar compound, but on the other hand is not very water-soluble So this is why we cannot inject any sample in 100% water because then some analytes will also precipitate out

So one of the tests we often do Is that we inject the samples to see if any you can actually see peaks disappearing By if you inject it too low organic, so so so all this analysis and screening is also compromise

Because you could also go in the other way and you can get your compound Precipitating ounces powder and then it has to redissolve and that's a time process that takes a lot of time Yeah, you can actually see this So this is actually some true samples I've made together with Lars Helgein

These are some rats that have lived by stuff that we were eating today and Then the only thing they have been allowed to drink is actually water with sugar in so we're looking at some lipids and First we looked at the samples and some of the analyze and this should be some special phospholipids

You see they look like really crap. It looks like there's a lot of isomers and you cannot resolve them And here you could say oh, I'll have one two three that could be more. This is terrible Then we actually owe This is the standard way of extracting them. So we will often end up in this mixture with chloroformethanol

to solve anything Problem is that then we have a postdoc and she evaporated these samples down Take took my mom in isopropanol and inject them again And now we get one nice peak here and if we look at the area, this is the intensity

But actually it turns out that all this fits well there so It is very important that we inject our samples, right? well, here's actually the target and It will stay in the chloroform droplets that will be formed in the column and they will

Be carried down for a while. And then this is why you get these to make things even more complex This Is an

Analyte and it likes to stick onto the whatever sits in the column also except the column face itself So this in this case, we had the instrument running the peak looked like a red curve look like shit or really really shit We took the same brand of column. It was the same batch everything new one into the system two blank runs

And sip this one through and now we get a nice peak So what happens here is probably some kind of ion exchange and and that there are some crap sitting inside the column And our analytes are sticking to that

this is why we also have to change the column and The worst thing was that was among our other analytes here. They were not affected at all So sometimes we have to look into the we have to have some very troublesome compounds to evaluate our LMFS

Okay, I Talked about before that when we mix things You know if we take some liquid in here and takes it out And we have a mixer inside. It takes a while. You had that first semester you have this classical Where the first order look at it make decay

So this is also what happens and this is why we want these very small mixes and everything technology wise but Anyway, we have to cope with this so so again This is the easy one. This is the one with the blue one here This is the one you just type into the computer and then you think this is what I get

The red one if you have a very modern system is probably what you get But you have an older system or a system where you just put in a bigger mixer you could have the purple here So it also means that you can think that you have two identical HPLC system in the lab and they may not be so

Also, another thing is that the scientific papers you read That this was done on a water security or a HLN 1100 system They're probably gonna write which mixer size they have So you cannot use their retention times even though you buy the same column because you don't know the delay of their gradients

so we call this is this is a really really important part also because When you go the other way You can program this one down, but you have a delay so what you type into a gradient table is not what you get and

We have another small assignment here. So let's say that You programmed it in and you say well, I just need two minutes for it to equilibrate

After the gradient and then I'm happy Then what you really get is this purple curve So you can also see we don't get down to the percent organic we think we get and What do you think happens if we inject it again here 20 minutes here and we are here. What would happen?

Will we have good retention of our analyte? No So in this case We'll have no retention So this can be very frustrating because you're coming into your HPLC system

You're putting on your solvents and everything you do the first run and you get a nice retention Of five minutes and you say well, this is perfect I do another one in the run just to test and suddenly you don't get the same retention time And that's because you do not have the equilibration of your system and

so what you need here is really perhaps to keep down your your system perhaps up to 10 minutes to really get your column equilibrated, right and Of course the lower the flow rate in your column the longer this takes

You could actually have seen this You recall the curve I showed you up the pressure of the system There you would see the pressure would not really have gone as high because you know when you have The pressure when you're here should be the same here

so In this situation you would have seen that I have a lower pressure because I actually have more a system nice file So looking at the pressure curves could also told you this but this is the most common error I see and I see it a lot Also, we're quite experienced people

So also to make sure you understand everything Can we make a gradient run with these solvent systems, let's just say one minute and then we'll continue

You didn't make a Yeah, let's go to yeah, you have some practical stuff that's right we take the detectors last

But before you go

Last time we talked about that. We're going to need a second exam day Hope you all check your calendars. We're going to try it with a poll We also know that a lot of people are not here today And you can also just tell us the old-fashioned way whether or not It's best for you that it will be on Monday the 14th or Wednesday the 16th

So we have the 15th also, that's the official exam day, so We'll try this and then of course just come and tell us if one of these Yeah, but we'll see and we'll try of course if some of you can only do the 15th, of course you'll be

Examinated on the 15th If anyone is capable of doing Also, I think three or four teams signed up for the practical exercise on next Monday and

If you want to have any kind of say in who you're going to be in the group with on The lab exercises on Monday, please send me an email It doesn't matter if you're only two people or you know, I don't know anyone on this course. I don't mind just send me an email and I'll set the teams together and

It will be ready on on Monday, but it will be nice for you to be in the lab with the same people As you're going to do the uh the big assignment with Yep But yeah vote for that whenever and then do the exercises and then

So from now on only exercises for today and then we'll take the detectors next time So we just wait for this one and then and we assume the rest will prefer the 15th

17 that will be the last exercise

And we will also upload the solutions so you can sit and look at them