Let’s begin lecture six. I’ve touched upon negative ion mass spectrometry. And I like to say there’s nothing negative about doing negative ions that could be dictated by the chemistry of the molecules that that’s the preferred way to go. There is an excellent reference that I just noticed I do not have in the reference list, I should add that in the future, that makes a strong point of how important negative IMS spectrometry can be. You can do it for electrospray or APCI, of course, as I’ve described, and the reason it has relevance is an acidic molecule such as a sulphonic acid is well suited to create a stable anion. If the molecule can be a stable ion ion, either in solution or in the gas phase, that’s a good candidate for negative ion detection. And the added benefit that I’ve mentioned a couple times is that many of the chemical interferences, the other molecules that can cause chemical noise, do not respond to negative ions. So you will not see them even though they’re the baseline is therefore often low lower, and the interference from other molecules is less because those molecules do not support or provide negative ion detection. So your molecule in the presence of a base in solution will create a deprotonated molecule that’s an anion. And if it’s in that one that’s in the gas phase, it can enter the our inlet system over a mass spectrometer and B mass analyzed. An example about how positive ion produces ADOX. Here’s the positive ion. Example, methyl paradeen. Remember, the proton affinity of a nitrogen is high, so you can readily protonate such a molecule we have a nose plus h, but we can also have n plus ammonium ion and then plus sodium ion. So addicts in the positive ion mode are quite common. But they also do exist to a lesser extent in the negative ion mode. There are different kinds of addicts. If you have ammonium acetate present in a low percentage in your mobile phase, and you’re in a negative ion mode, you very likely will see an M plus 59 minus your mot like your weight plus 59 will be an eye on that you see as an addict. There’s not a lot that you can do to control that is a function of the chemistry. The kinds of molecules that really produce a lot of atoms such as this are those that are neutral with oxygens in them steroids, sugars, carbohydrates, many oxygens in the molecule, they tend in the presence of ammonium acetate, or ammonium formate. to form these addicts. Long ago, we did some work at Cornell with lipids, which are many of them are non nonpolar. And we had a trace amount of chloroform, we were doing normal phase chromatography with a small amount of chloroform when we saw m plus 35. Minus there’s there’s trace HCl and chloroform, you don’t usually know about it, it’s not real surprising to see it, but that little bit of chloride ion in the chloroform that was also a small percentage in our mobile phase, we saw an M plus 35. So if you have a source of an anion, a bromide, a fluoride, two, primarily the chlorides are the ones that you see the most. So do not be disturbed. If you see a negative ion detection, some addicts there are fewer numbers of them. And they will be different than they are in a positive ion mode. So negative ion formation iron molecule reactions, we are doing gas phase chemistry. As we reviewed, if we have a molecule and have a basic anion present, you can remove the hydrogen if it’s acidic. So this applies to acidic compounds. Remember that an alcohol like ethanol or and butanol or any kind of alcohol is not a weak acid, only one that oh, he has an aromatic ring. So it’s a funnel that then is a is a weak acid. So for alcohols will not do this. But this is a typical strong organic acid or carboxylic acid, and he can readily remove in the presence of base a proton to give you the carboxylates anion. I happen to remember that I think the molecular weight of benzoic acid is 122. Until you get to 121 charge transfer, we saw an example a couple of examples that were you have an ions created in solution in the presence of that your molecule which is neutral, will accept the charge. So that’s charge transfer, or charge sometimes charge exchange, it’s called two different things. And that can occur nucleophilic addition, it’s what’s happening when you get that when you see that additionally, and the species here, the acetate ion the ducting to the this molecule, so that’s nucleophilic I feel like addition, and you can have nucleophilic displacement as much less common but you can actually with an eye and displace the A from this AB compound to be an anion. So again, that’s a form of trance charge transfer, but it’s caused by nucleophilic displacement. This happens generally in the gas phase and that spray poem. Well, those are four different ways and to produce anions and negative ion detection structures of nine drugs in a mixture. If you take a look at each of these, some of them are acids, this is endo indomethacin. And you can see it’s a carboxylic acid we look across here, that’s caffeine is not an acid albuterol is a phenyl. That’s, that’s an example of an alcohol. That’s not acidic. But when the O H is attached to the aromatic ring, as organic chemists know, that is a weak acid. And so you could do that by negative ion detection. And you take a look at each of these as another acid. This is reserve pain, which is basically an indole alkaloid that’s a basic compound. Cortisone is a neutral molecule. If you were to do this in the presence of ammonium acetate and a positive ion mode, you would look very likely C m plus 18 ammonium ion, if you were to reverse the polarity of your mass spectrometer to negative ion mode, mono acetate is still there, the acetate from ammonium acetate, what a duct to this has got 1234 Or five oxygens. It’s the multiple oxygens that that supports the addition of an acetate ion. This is diarist of estradiol, a growth promoter. These are all drugs, and they’re different classes of compounds. I circled indomethacin here, because we’re going to take a look at how it behaves. This is electrospray ionization total and currents for positive and negative LCMS. So it’s all electrospray. There’s nine compounds in there. And under positive anti AIC for electrospray. We see indomethacin here, that same sample injected under negative ion conditions. indomethacin is one student. Yes, I said used to say glows in the dark, significantly better sensitivity. So one of the benefits and the reasons to try negative ion is it may be very well give you a much better sensitivity for a particular molecule. So this is kind of eye opening to see the differences in response. I think we in the lab, there was a question or I raised the question, there were a number of compounds present, but you didn’t see them all. So there are the same nine drugs are in this mixture. But you see easily three and maybe four way or the rest of them, they’re there. But they do not respond to negative ion detection. They’re not acids. So you shouldn’t be surprised by that. So that’s a very good question. Very relevant question. The LC conditions, the mobile phase was changed to induce or to pill civitate negative ion, we had either ammonium acetate or an acid up here to be positive i We need a source of protons an attic. And that’s I’m really glad you asked that I was going to ask it later. But it’s better that you ask it. And it’s very perceptive of you, we need to appreciate that when we switch what we’re doing LCMS non infusion not something else, when we switch to negative ion, we may, we probably need to change the mobile phase and therefore the chromatographic conditions. to favor the ionization we want, we need to have a base presence. So this these are different LC conditions. I don’t see them actually shown here. We had a summer student here from IIT one year, and I met her through the mentored her through the summer to it wasn’t rocket science, it was simple LCMS. And these examples come from that we made equal molar mixture of these nine compounds. And in a perfect world, you’d like to have them all be the same peak height, when you do LCMS, you’d like to see nine peaks all the same height in an area. That’s a dream, you don’t that doesn’t happen in LCMS, the response drummer varies dramatically based upon the chemistry of the molecule. And other kinds of detectors. That’s not the case. And so in this case, clearly indomethacin is better done by negative ion if you want sensitivity. If you want to see more of those compounds, then you better be doing positive ion detection, you may be you need to do both of those experiments in sequence to see everything that’s present. So look at how much more sensitive indomethacin is in a negative ion mode. Positive or negative by electrospray. Here’s on the medicine, notice the Attucks here’s the unpolished H at 358. We see addicts, this is just a zooming in of the 357 358 you know the the isotopic abundance here is an inset showing you the details of the isotopes, but I want to focus on the X here on the positive ion mode but in the negative ion mode, we see very few virtually no addicts in in the negative ion mode. So in general there are fewer attics on the negative ion mode there are sambad in this case, fewer I also bring your attention this dimer we saw another example earlier when you inject too much sample it doesn’t not have If you do it optimally, don’t inject so much. But it will definitely happen if you inject too much sample, it interacts with itself in the gas phase, and combined is bound by a proton. So that’s a dimer of that molecule. positive ion formation on an atmospheric pressure come organization, it’s very important to realize that is not solution phase chemistry, the chromatography takes place with your analyte in solution, but we spray it into a hot plume, and in that plume is volatilize solvent, but also we want to volatilized your molecule in a very quick mode. And it’s in the presence of a lot of vapor. So the Leidenfrost effect is there, there’s some blanket of solvent around the molecule. But if it’s a particularly labile, fragile molecule, it can thermally decompose, but we do not protonate it and then unless it’s vaporized, neutral molecule, so reminding you what the heated nebulizer is pneumatic nebulizer with a corona discharge in the negative ion mode, we form this this, this dot minus if you will, and oxygen is key to that addition to the neutral molecule in the sequence of a gas phase chemistry takes place that ultimately gives you this species was rakes down to what you really want and what you actually see. And that’s in minus h minus ions are formed at this region right here, the heat creates the vapor the heat of autolyzed molecule, and when the ions form, they make a turn and go directly into the vacuum system, the initial vacuum system, but down here is the heated capillary that you’re adjusting the voltages to and heat yesterday. So here’s electrospray, versus APCI. The previous example was electrospray, and positive and negative, this is negative ion electrospray, versus negative ion APCI. And you can see it’s the same mixture of nine compounds, several of them are not seen because of what we mentioned before, so and didn’t know Matheson here that responds fairly well the negative that responds a little bit better but but fairly well also an in APCI mode. So comparing these two, either one, it looks like if negative ions is a choice you’re going to make either one would be suitable. These other molecules if you remember diaphysis, the best Ra was a DI phenyl weak acid, so you’re readily see that cortisone conform the attic with the acetate. Naproxen is an acid or carboxylic acid, and albuterol is also so that’s why we see these and do not see any of the others. positive negative mass spectra for indomethacin. Here is the m plus h 358. Without seeing the dimer in here, we see that there’s too much heat in the sprayer or the ion source, which can cause fragmentation that negative ion mode for this indomethacin was a lot of fragmentation here, a lot of experiments have been done in the past to differentiate is that due to the heat, or is it due to the ionization, I both of these API techniques are generally accepted to be very mild ionization techniques, they do not impart a lot of energy to the molecule, electron ionization and parts a lot of energy. If you remember, a typical carbon carbon bond is about 110 kilocalories fairly strong bond. And when we use AI, we use 70 electron volts. And electron volt is is a lot of energy. And it amounts to about 2200 kilocalories. So when we do AI, we’re hitting a molecule with a hammer, literally. That’s why it fragments so much in these experiments, it’s very gentle does not impart energy. That’s why people who would triple clouds use MSMS, you have them collide, the ions collide with occlusion gas, like a car going to a telephone pole. And something has to happen, the molecules fragment, we can do that as he did. In the lab, he described the INSOURCE Cid increasing the voltage to accelerate ions, I think that may be yet to come, we can make these ions fragment as if it was MSMS. It’s not really MSMS, because it’s not pre mass selection. But you can cause this fragmentation to occur in this case, so it’s not due to any MSMS due to too much heat. And as part of your method of development, as we discussed in the last lecture, it may include lowering the temperature too low. And also you don’t see this much fragmentation. If you’d suppose you were doing selected ion monitoring, you’re doing quantitative analysis, you want high sensitivity. And you want to monitor to this protonated molecule. This is a species, all the iron current is in that if you have these conditions, so you’re getting all this fragmentation. If you’re going to monitor just this ion and selected ion monitor, it makes perfect sense. But if you do that down here, you’re going to have much reduced sensitivity. All the ion currents spread out through all of these. So when you’re doing si M, you don’t want fragmentation occurring. You want all the energy that’s been used to make these product fragments, want them in the precursor Orion implies h. So as part of the method development, for high sensitivity LCMS quantitation, you want to make sure you do not have fragmentation occurring. And you do that while the full scan acquisition of the molecule, you might do it with repetitive injections by Fia, you might do it by infusion of a solution of your molecule. That’s really the ideal way of doing it. As long as you don’t contaminate your instrument. Without that molecule, it’s in there all weekend because you’re sprayed to high concentration. And so you can systematically lower temperatures and energies to preclude fragmentation that would be part of a method development, optimization. And other important area that probably is not of interest to folks or in the roles that you have. But I mentioned yesterday, ion chromatography or ion exchange chromatography. This is generally used in environmental applications. It’s used to look for inorganic ions and cat ions, cat ions and anions, as well as some organic catalyzed anions. There are commercial companies in this country that the big the big giant is dynamics dynamics, which was purchased by thermal power is the predominant vendor for ion chromatography. This unit is made in Europe, and it’s MetroCount met metrohm is the name of it. We have one of those systems here. But it’s an ion chromatograph, I won’t go into the details of the mechanism. It is a form of chromatography, but not for organic molecules and drugs. It’s useful for large proteins, small nucleotides, amino acids, inorganic ions, and inorganic cat ions. And I’ll show some examples here. So these think of the Masseys member, we often do not scan down below 100 or 150. And here we’ve got ions as low as 19 for fluoride. So this is a chromatographic profile for ion chromatography mass spectrometry, in this case, selected ion monitoring. What are you going to? How are you going to fragment a fluoride ion? Do you expect to see any fragmentation from the void No. And so you don’t need to do high energy or look at a full scan. So select that I am honored to even see I can’t quite tell the time axis here, six, six or seven minutes. But each of them is fairly well separated, perhaps with the exception of these two, but nitrate nitrite bromide phosphate, very nicely separated by Ion chromatography, you could not do that with a reverse phase column. These things go right through the column, and so the packing isn’t even there. So these are special columns and special chromatography. Here’s 10 parts per billion of fluoride in water, with a signal to noise ratio of 40 to one nice strong signal coming out in four minutes. This is the limited detection for perchlorate perchlorates, an organic anion that’s of concern and environment, large cities and industrial areas where this is in the drinking water or the environmental waters. And so you can easily do nicely do perchlorate. These are ions and solutions. So they’re not going to be retained by chromator conventional chromatography here that is diagnosed the best drawl. And this was an early part of my career in in New York state. There is a quite a cattle industry, agriculture and industry, a lot of cows and beef cows and so forth. And at the time, we’re not going to do very much detail there were this as an illegal drug. It was not allowed the USDA or the FDA. But some some of these drugs were coming in from Canada into Utica, New York, there was a slaughterhouse in Utica, New York. And this was being used by veal calf farmers, farmers to grow veal calves, the calves get to be just a few months old veal is a young cow as opposed to a fully mature cow. And the margins for or for success. Financial success are very narrow. If you give this kind of a drug to a veal calf, a week or 10 days before it’s slaughtered. It means a 20 to 30% increase in revenue to the farmer. And if the farmer has 200 of these animals as they gain in money, except that people in Europe, a few people in Europe and a couple of us died by eating an injection site of veal a piece of veal steak where the needle went in. And there was a high level of these compounds in that piece of meat and it killed some people. And so it was vehemently outlawed. And I was very involved in the early days of analyzing tissue, bovine tissue for diagnosis of Estoril. That’s all pretty much hopefully pass. I think the law tightened up on a lot of this. This happened back in the 80s. And it was quite a problem here and in the US. Also in UK. There has been some issues of people being told not to eat beef in the UK for things like this. But that’s a great content compound for negative ion detection. Daya soap is Tabasco. Let me ask Given an academic question that’s got two funnels, you could have vision have an NI and at each end, if you had were doing electrospray and had a Diana and you would have a a doubly charged ion. So would you expect you could see WHR Dionysus 268? I would look for something at 134. Do you think you can see that? If you did, would you predict as you’d see that in the mass spectrometer, you might in principle you could, but they’re so close together that they repel each other destabilizing. So one anion is more stable than two. So you generally do not see Dianna tons of small molecules like this, even though in principle, it could happen if you have a peptide with a long chain and the NI on one end can be a long way away from the other one, then you’re going to have W charged ions. So without I come to a list shortlist of relevant references on this material