what's up everybody professor tomney here and it is finally time for the next lecture in the hplc course and in today's lecture we're going to take a look at ion pair chromatography so i know people have been waiting for this lecture and we're going to get started right now okay so what are we going to cover in regards to hplc with ion pair chromatography so first of all we're going to talk about what is it and when is it appropriate to utilize ion pair chromatography compared to some of the other versions how does it actually work how can we manipulate retention time and optimize this method what mobile phases should be used during this type of separation and then finally what are some important limitations and or considerations when we attempt to utilize ion pair chromatography so if you have not already i would strongly encourage you to see the first three lectures in this series and the playlist link will be in the description box below if you go there it is very important to watch all of those but especially the third one in the series which is on reverse phase chromatography because that's going to be some background information for the ion pair chromatography moving forward in this lecture so make sure you go down to the description box and check those out if you're up to date that's great then get ready for this new lecture right here i also just want to mention really quick if you want to support the channel chemcomplete.com again down in the description box you'll find everything there i have guides that are available they're sort of like ebooks that i've written that cover content that i address on this channel in far more detail than the videos go into so it's a nice affordable way to support the channel if you like what i'm doing to keep up my work and i will mention i am in the process of writing my hplc guide so it's not available yet by the but by the time i finish this series on youtube and this uh entire course it will be available and that information will be down in the description as well all right so let's get started regarding ion pair chromatography in regards to hplc all right so the first thing you need to understand is that many times ions are going to need to be handled differently when we're using hplc so in the last lecture we learned about reverse phase chromatography as i mentioned a minute ago and reverse phase chromatography as a general reminder is a non-polar stationary phase or column and it uses a polar mobile phase right so we talked about sort of like the c18 columns uh which have these very long hydrophobic portions that are attached to the silica bedding and then we used various solvents primarily being water methanol acetonitrile and then sometimes tetrahydrofuran thf so those are polar solvents water and methanol being very polar and we talked about different gradients we can use with those okay well we're going to have a similar setup in ion exchange um but there's going to be something different because those columns given the non-polar stationary phase they don't do the greatest job when you have very very very polar analytes and ions or something that is in a charged form is going to be in that extreme realm of polarity okay so regular reverse phase hplc it works well for most neutral organic compounds that are of modest size and polarity but when we get to those ions it tends to skew the polarity to the extremes and then as a result we're really left with two choices and so one of the first questions is how do we handle these different ionized analytes that we are attempting to separate so maybe you're dealing with some amino acids that have side chains right and they might have different charges so we're really left with two different uh ways that we can approach this so number one we can attack we can attempt to adjust the ph okay now when we attempt to adjust the ph in a reverse phase setting what you're really trying to do here okay we can set this sort of equivalent to this is we're going to be attempting to suppress the ion behavior right so we could say suppress the ionized forms so by changing the ph you think right back to your chemistry knowledge and you've got this various ion that's sitting around primarily acid or base and what we can do is we can attempt to neutralize those ions by adjusting the ph of the solution so when we do this it can present two issues that we have to be careful with so this can work sometimes uh but we have to keep two things in mind so the first is that you may have a large number of ionized molecules that all have various pka levels and that's going to lead to a result where there's no single optimal ph for suppressing all the ions in solution okay so again a common example this would be amino acids that have all the various side chains right i've got some acid and i have some base related okay so the issue there is that if i have both acid and base ions present in solution or you could call them conjugates if you want i'm not going to find a single optimal ph that gets rid of the acidic ions as well as the basic ions right so i would need a high ph in one case and a low ph in the other i just can't obtain those both at the same time it's not going to be possible so that is one of the major issues okay when we have mixes of both weak acids and weak bases together it's going to present that sort of problem so what's the second issue the second issue that can arise with this technique is that it's only going to work for mildly acidic or basic compounds right so if you need a very strong acid or a very strong base to deal with these extreme ph levels that would be required then your pkas start to fall outside of that weekly acidic or weekly basic realm and you're going to need to change the acidic conditions to a extreme degree and the problem with that again if we go back to i believe it's the second lecture on columns is that we have columns that are going to be sensitive to extreme changes in ph and they can rapidly degrade the expensive hplc columns and ruin the effective separation techniques so remember we talked about if we get too extremely acidic or basic the disruption of the silica bedding and or stripping away the um the added material right so like for instance the c18 material that's added on the reverse phase you can strip that away or you could actually degrade the entire silica bed itself either way it's not ideal to be swinging to very very high or very very low levels of ph and that's because it jeopardizes the health of the column it can rapidly degrade and hplc columns are expensive we can't be going through them like that let alone the fact that it's not going to give you the effective separation okay so if you have only weak acid or only weak bases present and you can slightly adjust the ph without ruining the column this is a legitimate pathway now what happens if you are in one of those other situations and you don't really have the luxury or the ability to uh ex basically turn the ph way up or way down okay so the second option is to utilize a different hplc method and that hplc method is the topic of today you can use ion pairing hplc alright so then we're obviously going to get into ion pairing hplc and what is it okay so in ion pairing hplc a charged analyte will typically be added to the mobile phase and this is going to assist with the chromatographic separation so the analytes added usually have a hydrocarbon based portion that's going to interact with the stationary phase and then it'll also have an ionic portion which will pair with the ionic compounds that are to be separated so to give you a rough or a general idea here right here's the inside of the column and we've got the silica based bedding right down here i'm not going to draw it in great detail and then we've got sort of these long [Music] c18 right pieces of material that are coming off so the idea is if i want to do an ion pairing the ion pairing is going to have some hydrophobic portion because this right here is going to be able to interact with this right here so i need to have some sort of interaction with the actual phase that i'm going to be using and then on top of that it's going to have some sort of a ionic or a polar head so for example and we'll take a look at this in more detail in a minute okay maybe some sort of like a sulfate or sulfite okay something like this so you've got the long hydrophobic tail and then you've got a polar portion in front and that polar portion is going to be able to interact with okay whatever sort of charged analytes are coming through that you need to separate out from one another right those would be able to without changing the ph kind of interact with these but the benefit is this tail right here is going to have interactions with the columns so you're going to kind of almost take the silica base column which has been adjusted with c18 addition and you're going to adjust that even further by pairing up these ions with the column all right so that's the general premise on how this is going to work so i i do have some common agents that are listed here now these are um one of many different types that can be used but these are some of the most common so hexane sulfonic acid salts and it doesn't have to be hexane hexane is a common one but they also have the heptane and the octane sulfonic acids okay and we'll talk about what the difference between using some of those might be in a minute or two so you go here okay and i gotta make sure i get my counting right one two three four five six okay and then it would come to a head with the sulfur portion oops that should be a single bond okay so here's one type and this is the type in the example i tried to use above okay this would be one type of a ion pairing agent the other type that is commonly used is the quaternary alkyl amine salts and those are simply nitrogens and they've got four different alkyl groups well they're actually technically usually four of the same alkyl groups right um but the nitrogen has a plus charge on it so you notice these are both ions or forms of ions right that's what makes them ion pairing agents they're going to pair with another ion that's coming through that you want to separate out so what could r be for those that aren't as familiar with organic chemistry r is a term to just represent rest of the molecule so it could be something as simple as a methyl group okay an ethyl group and you could kind of go up from there um butyl is very common so for a butyl you would see a nitrogen and you have one two okay one two three four like that one two three four right and it goes around that way four one two three four like that okay so keep in mind if you have these hydrocarbons here's your hydrophobic portion and then the nitrogen with the plus charge would be the actual ionic portion okay so these are two of the common ones now um just to clarify here the sulfonic acid group is typically used when you're trying to separate base ions and when you have the coronary salts the amine salts those are more often used when trying to separate acidic ions and that just has to do with the general principle of what's left behind right so let's take an acid for example if i have an acid attached to some group and by its very nature the acid leaves and becomes h plus that leaves behind x minus whatever that x group might be right and so if i have x minus as the resulting acidic component then that's going to pair well with the plus in terms of ion exchange so just keep that in mind as far as the individual uses okay so how does this all actually work down at the chemical level inside the column we kind of talked about it a minute ago right so ion exchange is typically going to take advantage of the hydrophobic material that's present in a reverse phase hplc column so remember i drew the c18 material up here right that's certainly not 18 carbons long but the idea is that we're going to get interaction between these two hydrophobic portions for the ion pairing agent and the actual hydrophobic portion of the column for the reverse hplc setting okay so when these ion pairing reagents are introduced they have significant hydrophobic sections that'll be interacting and experience interactions with that stationary phase and it's going to significantly slow the illusion off of the column for the ion pairing agent okay so the result that you essentially have is that you've created a c18 column that's going to have a charged ionic site at the very tip of that c18 column facing the mobile phase flow so what's going to happen with that is that this will then act to slow the elution of any of the oppositely charged ions that would normally be so polar they would just bypass the hydrophobic c18 portions entirely and now they're going to slow down and have some interaction and that slowing effect is good that's what we need in order to actually get some retention time and have some proper illusion and a clean chromatogram where there's actual separation going on okay so how can we alter retention time in an attempt to optimize the resolution signals so that's one of the next things we want to talk about here one way that we can increase the retention factor is by changing the hydrophobic portion okay so if we and this is for the ion pairing agent so one way that we can significantly affect the retention time here is not charge change talking about charges change the hydrophobic portion so if you remember a minute ago i was talking about the fact that this was hexane up here and it doesn't always have to be hexane i could change this into an octane right and by doing so i'm going to increase those retention factors so for instance if i use an octane-based sulfonic acid instead of hexane it's going to increase the retention times okay now another way to change the retention times is to add more or less of the ion pairing agent so that's another one so whether it's octane or hexane form right just sticking with that example i can add more or less of the ion pairing agent and i'll explain why in a second here so again both of these options are going to manipulate retention time and that's one thing we're always interested in when we start looking at the initial chromatogram so we want to know how can i manipulate the conditions for the given hplc setup that i have if i want to kind of change some of the separations right so how does this one work the concentration of the ion pairing reagent is going to be critical to the retention time because the more of it that's present than the more ionic binding sites uh the separating compounds are going to experience okay and the longer they're going to spend on the column so the longer they spend on the column that's going to affect their retention time okay but this does have an upper limit so when it says more or less you can get to a point where more has become so much that you're going to potentially run into issues so there is an upper limit where a saturation point is going to be experienced and there's also a lower limit where there's not going to be enough ion pairing reagent present to effectively slow down these ions they're just going to kind of wash off the hydrophobic c18 column now keep in mind that if you also have non-charged neutral analytes that are being separated in your mixture and you increase ion pairing reagent that may crowd out the column that would selectively decrease the retention time for these reagents and it would give them less access to the c18 beneath the ion pairing molecules all right so what i'm saying here is this ion pairing method is used when you have ions present so if you go back and you look at an original reverse hplc setup it is effective at attempting to separate neutral compounds from one another once you do this ion pairing reagent technique the neutral compounds are not going to spend much time at all in fact they're going to behave almost how the ionic compounds did back in a regular c18 column okay and the reason for this is that your ion material is going to start pairing up with your c18 material and that's what how you're modifying the column and by modifying that column it means that the neutral compounds they really don't have a huge polar charge to them in comparison and so they kind of just bypass or don't spend a whole lot of time interacting with the column at that point so keep that in mind there's a balance here this is primarily used only as a technique when you have uh ionic compounds that need to be separated okay so it should be mentioned here now that i'm thinking of it that when attempting to increase the amount of ion pairing reagent you need to be careful not to over saturate the column so you as i mentioned a minute ago you can have too much okay and if this happens uh the issue is that the excess ion pairing reagent in the mobile phase that will neutralize the analyte is going to be separated okay so in other words you're gonna have the ion pairing reagent and if it's over saturated on the column and you're still flooding more of it then there's no spots left on the column for the ion pairing reagent so it's going to start binding to the analytes that you're actually interested in separating and it's going to neutralize them and then they become a neutral compound that just gets washed through or carried through the column okay so a visual might be helpful there in terms of what we're talking about here's the column okay and our ion pairing reagent right is like this now i'm just going to represent this to make it easy as a minus charge okay so we've got our column here right as we go etc and then we've got the long c18 portions okay now when we do this it's very important to realize okay that what's happening here is that we've got interaction with the hydrophobic portions right here so this is what's happening you put this in and this is what it does to your column so this being an approximate c18 column so what happens if i continue to increase the concentration of this the ion pairing reagent would some point it's going to come through here and it's not going to have the ability to bind to any part of the column or interact with it and so then let's say i increase the concentration this much and i sort of have some of these that are floating free in the column then i turn around and i send in the actual analyte that i'd like to separate well as this analyte enters the column the idea is that it's supposed to be interacting with this surface right here and the plus stops here for a little bit and then here for a little bit and then here for a little bit right and the mobile phase kind of moves it along and we get nice separation but if the concentration of the ion pairing reagent is too high this which is free floating in solution now some of the ion pairing agent is going to easily pair up with this and you're going to create a neutral compound the plus and the minus will neutralize one another well if i then have a neutral compound that is exposed to lots of negatively charged portions on the c18 column it's just going to travel right through and pop off the column so you're not going to get effective separation or retention in a case like that all right so this can be very problematic because you now have a fully modified column bed with ionic charges and you're attempting to separate neutral compounds okay so again as i mentioned a few minutes ago neutral compounds rapidly elute from the column bed and that is a issue with too much concentration there okay now in terms of mobile phase of choice you're usually going to need to stick to the more polar mobile phases here such as water and methanol mixtures especially water okay now the main reason for this and you can call that a buffer if you're adding salts into the water but the main reason for this is solubility issues okay so you need the ion pairing agent to readily be soluble on your column and in your mobile phase so as you start to decrease to some of those other solvents we talked about in reverse phase like acetone nitrile and most certainly thf then you're going to be reducing polarity of your mobile phase and therefore the solubility of the ion pairing reagent is going to suffer and you're not going to really have a proper setup okay so you need to make sure that the ion pairing reagent that you pick is going to be soluble in the mobile phase in order to properly distribute uh and effectively have your ion pairing separation going on okay now let's mention one of the things we wanted to wrap up with here because we're going past well past the 20 minute mark okay a couple downsides so we sort of mentioned one which is you have to be careful if the concentration is too high for the ion pairing reagent but what are some others okay the first is that it takes time to equilibrate your column with the ion paired buffer mobile phase um and that's going to be much longer than a regular reverse face setting so when you install your hplc column and you're getting ready you can't just put a little bit of mobile phase into it and then start adding your analytes or your samples all of a sudden you need to let the column kind of equilibrate and get used to that mobile phase over a period of time it is much less in reverse phase regular reverse phase than it is an ion pairing because you need time for these ion pairing reagents to settle in and kind of get that hydrophobic interaction so that you get the ionic portion facing where the mobile phase is going to be okay so that means you're going to need to elute the mobile phase that you've created with the uh ion pairing reagent for a longer period before your column is sort of primed for separation now there's no exact number that's officially agreed upon but most people that have been around hplc agree it's usually about 10 to 20 times as much mobile volume that must be pushed through the column to kind of acclimate it uh to the ion pairing reagent and then actually be ready to start separating analytes so the other major issue that comes in is during cleanup time so it is extremely difficult i would usually say it's impossible to flush a column of all the ion pairing reagent after the separation okay so in a regular reverse phase it's easy to change solvent phases you just wash them out effectively enough over time and you can set up a different sort of solvent gradient or different mobile phases and mixtures okay this is not true for the ion pairing once you add those ion analytes in there it's a bit different okay so once the column has been ion paired it is usually sort of resigned to that fate forever and it becomes an exclusive ion pairing column for that specific ion pairing reagent or setup okay so you're going to take a c18 column that is very versatile could be used in a lot of different reverse phase techniques and you're going to basically say this has now become an ion pairing column only and it will only use the specific ion pairing salt or reagent that i have initially used here you're kind of permanently fixating the column only to that fate okay so given the expense of columns in hplc upkeep it's just a factor that you want to consider um because if you're going to be doing lots of different hplc techniques and you're you keep throwing a c18 column that's perfectly clean or good uh with an ion pairing situation you're going to end up rapidly going through columns if you're constantly needing to change the ion pairs or use another c18 column for traditional reverse phase so keep that in mind so with all that being said i'm going to wrap up here because we're getting close to half an hour so that is ion pair chromatography as usual leave your comments i will address them as i have been doing in other parts of the lecture series i love interacting with you guys you have very very well thought out questions regarding hplc and i will attempt to get the next lecture up uh hopefully sometime within the next month we'll see how long it takes to kind of flesh out and create i'm thinking where i might go for the next one is normal phase so we've done reverse phase ion pair i'm thinking normal phase might be next a good topic but we'll see um other than that as always subscribe you'll get up to the date notifications when i release all of this sort of content you click the little bell and you'll get the instant notification as soon as it goes live and then finally as usual hit the subscribe button uh anytime you are subscribed and or i'm commenting uh hitting the like button that really helps the analytics for the channel and allows me to keep making great content so other than that check out chem complete make sure you're up to date on the hplc lecture links down in the description below thank you for learning with me everybody and i will see you in the next one you