So lecture seven, the last lecture or tomorrow these completely in the laboratory. This is on normal phase flash chromatography. It’s not obvious to me why normal phase chromatography is what is generally been used for flash. With all due respect memory, as I mentioned earlier that I started out as a synthetic organic chemist, synthetic organic chemist are not generally strong in analytical chemistry, which includes chromatography. So the original chromatography was normal phase, that’s when we reversed it. So I think they’re a little bit origins are from the past, and why they use normal phase chromatography. But historically, they’ve used that which means they use hexane. It’s one of the possible justifications as hexane is more volatile than methyl or then water, reverse phase. And you’re going to see that the flow rates are hundreds of mils per minute. So at some point, they have to get rid of all the solid they have to blow it off. And so a volatile sovereigns easy to get rid of the non volatile. So historically, normal phase chromatography has been used. And when you see here, flash, you should think preoperative column preoperative LLC, they’re prepping milligrams to grams of material, which is not at the analytical scale. And so they want to be sure that the peak that they’re collecting is what they think. And so in the recent past, mass spectrometry has been coupled with this approach to do that. And so there’s a mains that I’ll describe where the LC peak when it comes off, the UV detector sees it and triggers the mass spectrometer to start collecting. Otherwise, the mass spectrometer is sitting there not looking at anything, we’re going to switch the effluent from a column when the peak starts to show up, as indicated by UV detector, going to switch some of that to the mass spectrometer, so we know what it is. And that’s what the nature of what we’re doing here. More recently reversed phase chromatography has been introduced to this field. And people are starting to use that also. And I’ll close with that. So why combined flash with mass spectrometry? The simplest answer is to be sure what we’re collecting is what we want molecular weight and structural information. Mass spectrometry is well suited for good selectivity. It’s a good way to determine whether there are Co Co eluting components in the peak and LCP and you’ll see later that these are rather broad peaks, it’s large particle size is not UHPLC quality, it’s broad peaks that have large peak volumes. And so it’s possible to have another component colluding and you don’t know the difference. It’s buried underneath that peak, mass confirmation of the collected fractions to avoid offline TLC and work on other additional work. And certainly, if there’s isomers, involved isomeric mixtures, including chiral mixers that can be accomplished with the appropriate chromatography. So what is flash chromatography, very large inside diameter columns, remember, we’re focusing on analytical LCMS 2.1 millimeter ID columns, 2.1 millimeters, these are to to set to 10 centimeters, how much is 10 a column that’s inside diameters like that, you can drive a little car through a kid’s toy through it, big ID columns, large scale, if you will, the length of 10 to 30 centimeters 30 meter mobile phase, the flow rate, not 200 microliters per minute, but hundreds of milliliters per minute. So your your hat 500 milliliter bottle that you might have on your system is going to be a 10 gallon milk can I know some very large volume container, large volumes of solvents being used, solvent consumption is many liters per day. Not all bad. But that’s what you do when you do prep the chromatography is LC CMS compatible, not without total flow is no way we can swallow or accommodate 100 milliliters per minute, if you will. So we’re going to have an arrangement where when that peak comes off, we’re going to sample just a little bit of that flow, a tiny amount of cut from that flow. So mass spec versus other flash detectors or these others have in use ultraviolet, we’ve certainly know a fair amount about that response varies by the compound. And by the way, the compound better have a UV chromophore, a carbonyl carbon, a UV, a double bond, some sort of a chromophore or it doesn’t see it, it’s transparent. Not all compounds can be detected in it’s not as selective as some other detectors. There’s another detector you may or may not know about called LSD of aperitive light scattering detector. This measures photons scattered from semi and nonvolatile particles that have been dried of the mobile phase through evaporation. So you’re doing LC you’re in a way your flash volatilizing but instead of a mass spectrometer, you have this LC el el Less D, it’s more universal than UV, it doesn’t need to have a chromophore. It just needs to be a particle. It’s more consistent response than UV, so better direct relative abundance comparison, but it’s still not very selective at all any particle will be detected by that technique. And finally, mass spectrometry is applicable to a large broad range of organic compounds, as well as providing chromatographic data, it can provide the identification or confirmation of the compound. And mass spectrometry has a broad range of utility. But it’s not really truly universal, there are some compounds we cannot see if we cannot make an ion because of its chemistry, then we cannot see it. Ion sources, no API source is truly universal. Seeing all things equally, the selection depends upon the compound to be analyzed its structure and the ionization modes available. We know about the common historical sources of electron ionization and categorization, but they can only be used with a gas source like GC. And it’s hard to put grams of sample onto a capillary GC column for there used to be preoperative gas chromatography, it may still be done. But I it’s not very common. Gas chromatography is limited to a small population of the total organic molecule space, I once one around ask asking a question to three or four of the top chromatography that I knew in the world. And I asked them in terms of the total organic molecule space that includes methane to proteins, any molecule with a carbon in it, considering all those and by the way, you can derivatized to your heart’s content. In terms of gas chromatography, what percentage of that total population of molecules is amenable to gas chromatography? In other words, you can inject it, it’ll go through the column and be detected. And the general answer is about 20%. Not a large population. When LC came along, it took care of the rest. In fact, some of those that are really difficult to do by in that 20% can be easily done by LCMS. So you can see the origins of my press my, my precedent, my preference for or favoritism towards LC ms, it’s really amenable to a large number of molecules, although not all, liquid introduction sources, analyze over 80% of these compounds, including POLAR NONPOLAR biologics, meeting proteins and peptides, as well as some polymers. Using a variety of ionization techniques in our toolbox, including these that we’ve talked about. We’ve not talked very much about dark but you have seen ASAP API as atmospheric pressure photo ionization shining a xenon light on it, we do not offer that as a source it has been commercially available by Agilent, and one or two others, but it has fallen out of favor it it works, but it’s not convincing enough or successful enough, it’s not better enough than something else, to have people still aggressively buy it. I actually do not know I believe it’s still available if you really want one from from perhaps Sykes or Agilent. But I haven’t seen one or heard of one being sold in quite some time. The other techniques work equally well, that are some benefits, but they’re not compelling benefits to get people to buy it. So remember these ion sources that we’ve discussed this dundun but we have the atmospheric pressure colonization source, that is typically used for nonpolar compounds and not real high molecular weight, you must not try to do a protein or peptide or an industrial polymer that we have to have a vapor pressure and that it has ionization initiated by the corona discharge needle. In comparison, though, or the other tool is electrospray ionization, which is typically pneumatically assisted electrospray as I describe, which is very amenable to very polar compounds, any molecular weight because of multiple charging, the mass range of the mass spectrometer most all of them are no more than what we have the event mass spectrometer is out there with exception of time of flight. Time of Flight has an infinite mass range, if you can make an ion that has a singly charged ion, that mast 50,000 You could see it at a time of flight except in the no ionization really does it electrospray makes multi charges, which remember increases the Z of M over z and that slides the envelopes down into a regular mass range. And so most quadruples are now currently 2000 Like your weight, mass range. And so we can handle those kinds of molecules. Not necessarily because of the mass range the instrument but because of the benefits of multiple charging with electrospray. So which one of these interfaces was first to be commercially just a trivial task? I think I mentioned this yesterday. Which one was firstly, first commercially realized? I’m not talking about homemade devices like Leo. So who’s in any idea what Your that was remember what I said? I mean, this is not 2018 Was it 1990 was a 1985 The first one was actually this heated nebulizer APCI. The fundamental work was done in 1971. Before I had any involvement in this had Baylor University by a husband and wife team, Marjorie and Evan Horning, they did fundamental studies of APCI. At that time, very early on, they did not use a discharge needle, they use 60. Nickel 63, which is a radioactive emitter wouldn’t be very handy in most of our laboratories. And the first commercial version of that was a 1979 by sciex. And in 1984, is when electrospray was really brought forward by John fen and a bunch of us worked on it and the commercial availability that came about in about 1988 or seven something like that, just trivial details on the side. So APCI and electrospray, generally ionized by proton transfer, accepting of a proton or removal of a proton. This the first being with a base, the second being an organic acid ionization may occur by forming attics with other species and you’ve seen examples of ammonium adduction 18 plus 23 is sodium plus 39 is potassium plus 33. Is Andrea knows is now methanol and so forth the CNI trail is 42 a mass molecular weight of a sila trills, 41, proton bomb dimer is 42. So electrospray and APCR are good for most drugs, metabolites, aromatic compounds, large number of these kinds of compounds. APCI is best for generally small less than 1000, molecular weight, volatile polar and neutral, not real polar, but neutral compounds like steroids. And we need electrospray for these more challenging molecules, RNA peptides, proteins, sugars, carbohydrates, and other classes of compounds. Why is this APCI preferred for flash. It’s simpler mass spectra, the organic chemists these are synthetic chemists that have made a lot of this compound, they want to collect it. So the purpose is to profitably collect, isolate from the rest of the reaction mixture, large quantities, grams or kilograms of those molecules. And so they want something simple. They don’t want a complex mess aspect from to interpret. This technique is less prone to attics and fragments and dimers and so forth. Remember that proton bound dimers were seen with electrospray? We generally do not see that with APCI. Why might that be? Why might we not see proton bone dimers you inject a lot of sample. And with electrospray, you’ll see a proton bone dimer is the heat of APCI. Those proton bone dimers are not covalent bonds, they’re weak associations. They’re easily dissociated by heat. And so like thermal spray, no APCI has this heat and generally dissociates and they probably are they may be forming but we don’t see them probably because a disassociation by heat APCI is generally easy to do. Less solid dependence as ionization occurs in the gas phase, less matrix suppression these are reasons to prefer or lean towards APCI. You could do you can do this flash chromatography mass spectrometry with electrospray. But the trend is to use this technique. However, samples must be sufficiently vulnerable, the balance will be vaporized and thermally stable in the temperature regime of these temperatures. I showed this before the chemical space of what APCI covers it’s generally this range of molecules in here, polarity being on the x axis, and molecular weight being on the Y axis. So it nicely fits in this region here. And this is where organic chemists synthesized compounds. So it fits in there nicely. What are the solvent considerations? solvents that are compatible with aqueous APCI and electrospray? Include methanol, C nitrile, and a little bit of isopropanol. Are there any solvents that you really should not use or cannot use? I mentioned THF tetrahydrofuran. That’s often used chromatographically by industrial polymers, that synthesis of industrial polymers, the columns they use are generally not reverse phase. And THF is a favorite solvent that can be used. It’s not real friendly. It’s higher boiling point, the methanol seemed natural, but it can be used. So that’s hard to imagine a common solvent that you should not use. chloroform can be used. It’s a heavy solid. Remember, it’s heavier than water, and immiscible with water. So there’s not any gotchas necessarily that I can think of. I did mention hexane be inflammable. You better make sure there’s no oxygen present when you’re volatilizing hexane in the presence of a spark sample countertrade Above 10 micrograms per mil increases the formation of cluster ions and may cause mastercharge intensity ratios to be unreliable. In other words, if you saturate them at saturate the signal the mass spectrometer, I think we saw a little bit of that yesterday in the group with Li, where the FIA peak looked like a chicken head that kind of detector goes up and comes down and then goes back up again you can saturate the detector, but you can also cause these dimers but you can saturate the detection system of the system. So less sample can be can be better. samples must be free of non volatile additives like EDTA phosphate buffers, SDS, Triton x, these are not friendly, LCMS friendly species, additives and buffers compatible electrospray and include the ones we’ve discussed before small acids, formic acid, and the favorite percentages, the point 1% or less, some cases even point oh 1% just a trace. more is not better. So here’s the situation that is the schematic setup. To do flash chromatography, Ms. Let me walk you through this. Here’s a giant chromatograph typically pumping at 10 mils per minute or 100 mils per minute, not 100 microliters per minute. And there’s a line a signal a wire going back and forth. So these two are talking the mass spectrometer, as it’s pumping, it’s going through a bow. This is a special valve, it’s called an MRA belt, mass rate attenuator it’s available from realigned is not ours. It’s a rapid switching Bell. The thing is obnoxious when it’s going, it’s going back and forth all the time, like this up here, click click, click, click, I’d like to put in a box and keep it quiet like quiet laboratory, but it’s actually switching. So why is it doing that, meanwhile, as this is going is going back and forth through this loop, and inside this box is the UV detector. So it’s a UV detector and a chromatograph LC column in the same box. And it’s normally running the effluent from a column through the detector and to waste a collection vial. But we’re going to put in series with that line. This this valve. And by the way down here, we’re going to have a delivery pump us SSI, that’s a manufacturer isocratic pump that’s going to deliver a CDI trail or isopropanol, or ethanol, with point one perform percent formic acid through this valve and to the mass spectrometer. So most the time two separate things are happening independently of each other. This LC is pumping 100 mils per minute going through the UV, and all of a sudden, and while that’s happening, the isocratic pump is pumping this mixture of sovereigns through the mass spectrometer, and it’s sitting there monitoring baseline monitoring nothing. But all of a sudden, through this wire, this red wire, the UV detector says hey, a peak is coming off. And that sends a signal to this thing to switch this valve, this MRA valve. So let’s look inside this valve. During this sequence as shown here, the liquid is coming into the valve here and going through this little line and back out of the valve to that detector. But when that UV signal says hey, there’s a peak coming out, This switches the MRA Bell switches so that the liquid is going this way now goes this way to the mass spectrometer. And it does that for a fraction of a second, it’s literally going like that. In fact, later it shows a 13,000 to one ratio is taking a tiny little sip of that 100 mils per minute, because we don’t want the 100 mils per minute going to the instrument the mass spectrometer, what’s going to the mass spectrometer is quite happy at 100 microliters per minute. So by sampling sweeping this loop with this one second aliquot, let’s say of this high flow that goes the mass spectrometer that goes back and forth like this. And can you imagine what the T IC looks like from the mass spectrometer, it’s got sample it’s got not got sample, it’s got samples not got sample, it’s a it’s a ragged peak, but we have software that’ll smooth that out and make it look pretty for you and I’ll show that in a minute. So the understand that concept, continuous LLC, too much to go through the mass spectrometer will want to peak shows up as indicated by the UV detector. The mass spectrometer says I’m here waiting, and it switches up valves and sends a little bursts of the mass spectrometer and we will get a chromatogram of that. So here are the experimental conditions for that. I love these these values, we’re going to look at three Thalys not rocket science, just three simple molecules, and we got a solution. That’s one mil viam. And down here we’re going to inject a half a mil half mil injection. Normally it’s one to 10 microliters we’re injecting and so these these the sample has 1.2 grams per mil not micrograms per mil or nanograms per mil but grams per mil, there’s three of them, and so on we inject a half mil we’re essentially injecting loading a half a gram of each of these compounds because we want to preferably collect it. We want three vials that have pure individual satellites in them. Here’s the radiant conditions for hexane ethyl acetate and in this case, actually, it’s not really a graded Meiburg most most the time it’s isocratic. So the MRA Valve has setting and we use a setting of 38. And that means it’s the 13,300 to one transfer rate one and a half micro liter per minute. And so that continually switches back and forth and makes a fair amount of noise because it’s doing that an actuator. And that effectively is ultimately sampling from that high flow rate. I’ll remind you what these valves are down here. This is not in your manual, I just added this. But when we are doing a switching, we’re going from this position to this position. What’s the difference the samples returning the UV detector. In this situation, the sample is sent a small amount to the mass spectrometer Does that all make sense? Follow that. So here’s what you get a chromatogram. Though the three valleys the orange, orange trace T IC, is the THC from the mass spectrometer, the UV traces the purple trace, it looks a little better. The retention times are mirrored, if you will. These are the orange ones are not too ratty or noisy. You’ll see some others that are worse, probably because a large sample size of quantities here. Here is the T AIC here’s the extracted I incur profile for each of these, these are broad peaks, they’re point eight minutes, 48 seconds wide, if you will, these are the protonated molecules for each of them. So this can be done. This is what is done to do preoperative flash chromatography. Here is a normal phase flash analog output from from that system, the organic trace being in the T IC. And this is an eight minutes and not too long to run. This is what more commonly it looks like though, the T IC looks noisy like this, partly because it’s going like this as part of the time there’s no sample going to the mass spectrometer. And so AV Yan has the only mass spectrometry software for smoothing such a trace. Normally, I don’t like the smooth, because if you are normally when you have unstable ion current as a problem, like a spinning sprayer, or a restriction of some point, but in this case, it’s not the mass spectrometers fault. And we do this just for aesthetics, mainly, and if you’re going to do any quantitation, it helps to get accurate areas on these peaks. So here it would be the typical setup. For the analog output for the Fraction collector. I won’t dwell on these details, but you should becoming familiar with how we fill in these boxes to tell the mass spectrometer how to operate when it when it goes. Here’s the three compounds again, the analog output is smooth to provide less noisy peaks. And here are the data after the smoothing come before these are the chicken heads I’ve mentioned the often noisy at the top of the peak. That’s all by normal phase chromatography. More recently reversed phase large columns have been available. And so that can be amenable to other kinds of compounds. And here’s 50 milligrams per mil not a gram per mil of caffeine sulfa dot methoxy friends of yours that you may be looking at in the laboratory, the column is commercially available pure flash column, that’s the name of the column, the conditions and the parameters are here, the MRA valve is 11. It’s not quite as rate a difference mainly because the flow rates not so fast. And the amount of sample is not so large value and makeup is similar to what it was before. And so these are the conditions for reversed phase flash chromatography. And here you get a nice chromatogram. Again, the purple is the 254 wavelength the UV, the orange traces the T IC. So why do you suppose this UV detector shows a great big P for the same sample that the mass spectrometer receives and that signal is quite weak? Should you be should you worry about this? Is this a problem? Not really, it’s detector difference response. This compound has a huge chromophore the UV detector really sees it. Whereas the mass spectrometer, ionization it has proton affinity is picking up a proton but it’s not a bright molecule, it doesn’t respond that well, all molecules have a different response and the better the response, the better sensitivity. But here’s kind of a dramatic difference in the response to these two. If we look at this peak, the UV and the mass spec are closer together. And that’s because they behave similarly. So that’s not something we’re about you should appreciate that just different detectors. And again the reverse face flash. A total anchor and profile for these compounds are shown here. Two of them are just barely resolved the selfie diamond Hochstein itself and own but in each case on iced tea AIC and extract Nyan current and with this information, this synthetic chemists can be really confident that the peak is what he or she thinks it is. Finally, the near last slide, this is what such a system looks like. The mass spectrometer over here the MRA Val, the Fraction collector. That’s another thing after the mass spectrometer when it goes the sample cones off the column through the UV detector, it’s collected. There has to be a Fraction collector and we keep them often most injections are made and each injection is collected in the same test tube or sometimes Erlenmeyer flask because the volumes are quite large. And so we have quite a number of customers in the pharmaceutical industry in in pharma where the NCS remember those are new chemical entities that want to be drugged someday after $15 billion to put it on the market. These synthetic chemists are called medicinal chemists. I never I always kind of smile at that they’re synthetic chemists, but they’re medicinal chemists. When they go to work and more pharmaceutical company, and they’re our best customer for doing this. They are the ones that do flash chromatography. So with that, you’re we’re not done but you’re done listening to me, which can give you some relief way. I want to personally thank you for taking this course. And hopefully go back as real experts with your instrument.