hello everybody welcome to another chem complete episode and what i would like to address today is an addition or an add-on to the hplc series so if you've been following the series you know we've been going over some of the major methods for hplc such as normal phase reverse phase ion and we've also discussed in great detail column chemistry as well as the basics of hplc and hplc instrumentation so because this is kind of an introductory course um with free content that i'm putting out there i did want to have a brief discussion on band broadening so that's part of what i would like to take a look at today in this lecture i don't know if this one will be quite as long as some of the other lectures that i've put together regarding hplc but we are going to talk about what is band broadening why is it a problem in particular to chemists that are using hplc what factors contribute to it and there's actually an equation we're going to take a look at and that will help us kind of understand mathematically what may be going on there in a general overall sense and then finally we obviously want to know what can we do to minimize these effects and sometimes the minimization can be a mixture of different moving pieces and sometimes it's just that the column is worn out itself and you may need to replace your column so we'll talk about all of that starting right now [Music] so before i get too far into the content remember you can always visit us over at chemcomplete.com and there you can pick up guides and resources hplc included over in the guide section that's a great way to support the channel you can also find some free resources over there and we will be expanding our site uh throughout the year this year as we continue to put out more free resources as well as paid for resources um you can find all the information over there to support us and as always commenting interacting dropping a like if you find this information helpful is always a great way to show appreciation for the channel so make sure you hit subscribe if you're not subscribed and let's get started alright so what is band broadening band broadening is a phenomenon when what should be really distinct separations and sharp peaks on your chromatogram are going to start broadening out or flattening and sometimes this can get very bad because you end up having these broadenings that are sort of increasing and pushing against other peaks and so sometimes you can get peaks that are almost merging together due to broadening now broadening is not the only thing that could be responsible for that you may have poor separation conditions and you need to kind of increase the retention time between those two peaks but broadening can lead to uh issues similar to that so when you see broadening if you were to take a look at a normal peak you'd probably come across and see hopefully something on your chromatogram that's somewhat sharp and distinct it's not a single line so there is some result of very minor broadening as you don't have one individual signal right and that's because as we load content onto an hplc column the hope is that the analytes initially start as this narrow band of material as they go down the column but they are going to kind of widen out a little bit due to factors that we'll talk about here and so what happens is not all of the content hits the detector within a very short period right ideally that's what would happen but we do kind of see a little bit of extenuation there as some natural processes tend to occur now obviously this becomes more and more problematic the further and further this gap becomes so if we're traveling traveling through the column here right and this is the analyte of interest that we're looking at the further apart or the wider the band kind of diffuses the more problematic the broadening can become and so then you may end up starting to see okay stuff that looks like this instead of a nice sharp peak and you get broadening of the band both in the chromatogram printout and that's really a reflection of what's going on inside the column your actual band of analyte that should be migrating across the column right is kind of flattening or spreading or diffusing out and causing this problem here so what is really the issue outside of the fact that the peaks occasionally may run together what is the problem here okay well number one when you're using hplc to purify mixture of components these analytes will often come out mixed together so that is not good okay if you start having broadening in other words let's say that it should look something like this right and they're kind of close together but they're distinct separate peaks and then you start getting broadening happening right where you've got one and then the other one before it really comes back down starts to shoulder off of it like this okay this is far more ideal this scenario over here versus this one right especially if we're not kind of returning to that baseline or as close to the baseline as possible and so one of the big issues as i just said is when you're trying to purify the analytes you could end up having the analytes mixed together okay so analyte mixing and that's obviously an issue because when we're doing chromatography the general goal of chromatography is analysis but it's also purification it's separation of materials and analytes from one another so the concept that we're now going to uh have to wrestle with is are the analytes separated well enough or are they starting to bleed together some right and it doesn't mean because normally what will happen is if you're interested in purification you'll collect what's called a fraction and so there may be certain fractions let's say you collect some right here that's some of the analyte you collect another fraction from here to here right maybe it's one every minute or every 30 seconds you're collecting a fraction right but at some point when you get to this area right here you're going to have fractions that are mixed together so you're not going to be able to get true purification and kind of maximizing your purified compound or analyte coming out you're going to have some that inevitably starts mixing together if this shouldering issue is occurring right and so number two one of the other issues here whether there's kind of a crossover between analytes or not is it could potentially lead to the in ability to quantify your results properly okay so inability to and i'm just going to put quantify here so remember quantify means to use numbers and so if we're talking about okay the broadening effect here it may lead to issues where when you're trying to analyze your individual analytes uh that have been broadening to a severe extent you're not really going to be able to quantify them properly relative to other analytes in the mixture and that's a problem not necessarily so much about purification but about analysis and quantification being able to say hey this mixture is 22 of this analyte right if something is sent in and you're kind of analyzing it based off of standards that you're making and putting into your hplc system so those are the two major problems or issues that typically can uh come across right so when band broadening occurs it is important to keep in mind because we're going to start talking about these issues here band broadening will occur naturally as a column ages so your stationary phase your column the more runs you put through it and certainly the harsher the conditions for those runs or the longer the runs take you are going to wear the column down you're going to wear the stationary phase down and you can end up having issues so if playing or tinkering around with these things that we're about to talk about these different factors don't seem to work then there is a probability that you may need a new column okay so i don't want to rule that out and think oh just for saving money we'll just constantly keep playing with these other factors it may be that you need to replace your column especially if you've had it running for a while okay however that being said there are several ways that we can attempt to minimize band broadening so let's study the factors that can play into it outside of just an aged column and then we can talk about minimizing those factors so let's get started with that by introducing an equation first okay so the equation is going to be referenced or spoken of as the van diemter equation okay so the van diemeter equation is a way that we can kind of tie together band broadening in a mathematical sense and there are four variables that play into the broadening effect okay so what we can do is we can say h is going to generally represent the broadening effect so the larger h becomes the greater the broadening effects are going to be equals a plus b divided by u plus c times u and this is our equation here that's going to help explain the broadening effect okay so obviously the next question we have is well what is a b c and u right because that's all going to affect h and h is a reference to height involving theoretical plates and how much broadening is going to occur but what are these other individual factors right so there's three of them and we will list them here starting with a and we'll move forward okay so the first one is something that is known as multi-path diffusion and multi-path diffusion is sometimes referred to as eddy diffusion okay so i'm going to put that in parentheses here and that's spelled e d d y so you may very commonly come across this referenced as eddie diffusion and this is a this is component a in the uh equation here and we will discuss what that is after we list all of these so i'm going to list b c and u and then we'll go into a detailed discussion what is multi-path diffusion okay so let's go to number two so number two on the list b is what we would call longitudinal diffusion now longitudinal diffusion is really you could say random diffusion natural diffusion this is a process that is going to occur throughout nature okay so when you have something that is in a high concentration in one area the tendency is for it to spread out to areas of lower concentration that is longitudinal diffusion and so that's the second factor and then factor number three is going to be what we would refer to as mass transfer okay and again we're going to go into more details here in a minute the mass that we're referring to here is the analyte and how often it's transferring back and forth between the individual particles in the stationary phase so keep in mind if you looked at our lecture on column chemistry you know that there are little small individual particles packed in most of the columns now a lot of times they have some sort of addition or layer on top of them right in order to provide better separation like a c18 column or something but when you look at the bottom there are still these particles these little beads if you want to call them that right that are there and so the question is how quickly or how rapidly is the analyte mass transferring particle to particle and is it all the same because it turns out that it's not always the same and we'll discuss why uh in another minute here okay and then number four which is the last one this is you on the list there and that is going to be what we would best refer to as the linear flow rate okay so one of the big terms that we always talk about in hplc is flow rate and that is referring to okay how quickly are we pumping the mobile phase through the stationary phase at what rate and we're usually talking here about a amount of volume per some time so maybe we're talking about you know five milliliters per minute or two milliliters per minute uh or whatever the the given unit may be we're talking about the flow rate of the mobile phase through the column and this is going to be a bit trickier we're going to tie it in at the end because it turns out that flow rates you can see in the equation here okay a plus b plus c if a b or c all increase or get larger then they're going to increase the broadening but you can see there's an inverse relationship uh between flow rate with b versus c and so that is to say if you have a low flow rate for u then that means you're going to have a tiny denominator and b is going to increase but it'll keep c relatively low and then if you allow the flow rate to increase or get larger it's going to amplify c the mass transfer but it's going to help minimize or shrink down b so there's going to be a balance here there's no perfect answer and you have to keep that in mind that when you kind of use the cliche term in moderation right this may be a case where you have to think in moderation the flow rate may need to be moderate so that we're not kind of maximizing or capitalizing on either of the factors longitudinal diffusion or mass transfer so keep that kind of tucked in the back your mind as we're walking through this and talking about it all right so let's start with a discussion on multi-path diffusion so multi-path diffusion is really talking about when you have a column and in that column you've got your various packing material okay and i'm kind of zooming in on this here right so this is your packing material your beads for the stationary phase that are here if you were to look analytes that are coming through in a band could take different pathways right so you could have an analyte that's coming through that kind of goes through the top here you could have another one that's coming through that goes underneath and then above this area comes out here goes all the way underneath here right and then arrives here you could have another one let's change the color just so it's a little bit easier to see here we'll use blue here okay that could start out right and the idea is that this is all the band that's uh initially loaded here right and this one decides that it's going underneath here then it's going to travel over top then it's going to go right in between here right it's going to come up right over here down and then finish through here okay so i think you can probably see why this is called multi-path diffusion it's because when we take a stationary phase and we attempt to pack the particles in there for separation there is the potential that there could be multiple paths that these analytes can take and if they take multiple paths they're going to be crossing over different amounts of surface area and that as a result means that you could have some broadening as they're all trying to make their way through right so imagine that some people if you're kind of racing around let's say a classroom or a building and you've got a lot of obstacles in the way chairs tables some people can run straight through the middle aisle to the back wall other people have to weave in and out of those chairs and those tables you're not all necessarily going to hit the wall at the same time right so you're gonna have some separation you're gonna have some people that are finishing before others what's the same is true of the analytes here when you have multiple pathways and the more pathways you have the more of this diffusion can happen the greater a is going to increase here okay now it's important to note that in theory a could be reduced to zero and could not contribute but the theory behind that is that you would need a tubular column that really doesn't have any type of packing in it and so that sort of defeats the purpose because we pack our columns to give additional surface area and the chance to add these additional layers right or modifications on top for better separation so it's a bit counter-intuitive to say oh i'm going to do chromatography and i'm really just going to kind of let these guys separate out without a whole lot of packed material in there or any sort of intermolecular force interactions that are going on on the column so in theory a could drop out but in reality you're probably going to be dealing with the multi-path diffusion factor to some extent all right so that leads us to b which is number two on the list the longitudinal diffusion right now as i stated before longitudinal diffusion is a natural process in which you're going to have high concentrations of analyte particles that are just simply going to statistically spread out and move from areas of high concentration to low concentration over a period of time okay and this can often times be a result of excessive time on the column okay so we're talking about b here the longitudinal diffusion uh in this equation so if there is excess excuse me if there is excess time and again this is a relative term right on the column this can become problematic and that is because this process is going to occur naturally so the more time that you allow a band of analyte to spend on the column the more the longitudinal diffusion can take hold and start moving about so unlike the multipath diffusion which in theory again it's very theoretical could drop to zero the longitudinal diffusion may not drop to zero because the very laws of thermodynamics in the way that concentration uh is going to spread with analyte particles would kind of be violated if you could somehow get this to zero so again the idea is that right we've got a band of analyte these particles are highly concentrated in one area right so this kind of represents our band of analyte if each of these was an analyte particle right and here's our column and they're moving along well the more time that they spend they're not going to want to stay all together in this area they're naturally after some time t going to be more diffused and spread out from one another now that doesn't mean they're spread out over the entire column right but it does mean that this band that was once very tight and narrow is going to start to broaden out and the more time that i allow that material to spend on the column the more the longitudinal diffusion is going to occur right now there's kind of a limit to this in the fact that if it spreads out it's still going to be interacting with the stationary phase and so separation will still occur to some effect it's going to kind of move as a group based on its retention time and its intermolecular interactions with the stationary phase but having really long run times can be problematic here okay so you're going to end up getting that diffusion and it's going to naturally start broadening uh if there's too much time that's being spent on the column and you do have to watch flow rates here so when we start talking about your flow rate okay and we'll talk about this more in the how do we minimize section but if you go back up here and you look having flow rates that are relatively small which means a low flow rate okay is going to increase this number so what does that mean let's think about that logically if my flow rate is very tiny let's say it's one milliliter per minute or half a milliliter per minute okay if this is a tiny number then b is going to balloon up it's going to get larger in its factor and that makes sense because what happens is if i keep a very low flow rate it's going to take a long time to flow or push material through the column right if i'm only pushing one milliliter a minute through it may take a whole lot of time until i get to this analyte whereas let's say that i'm setting it at three milliliters four milliliters per minute well then i'm having a bit of a quicker movement over the stationary phase with the mobile phase and so there's not as much natural time for this longitudinal diffusion to take effect and so it's going to shrink that b term okay so the time on the column and as a direct result of that having keeping very low flow rates can be problematic there and we'll kind of summarize that up as we continue to move along here okay so let's talk about the mass transfer so this is c on our list number three and when we start talking about mass transfer this factor is going to relate to the fact that all of those particles that we pack into the stationary phase so the actual be it silica particles or whatever stationary particles you're putting on there that may have modifications those individual particles or beads are generally going to be porous and the reason that they're going to be porous okay is because that will increase the surface area so one of the things that we want in order to get good separation from analyte to other analyte not within the group of analytes themselves okay is we want good surface area because if we have decent surface area there's going to be more opportunity for the intermolecular interactions and that means that hopefully we can get some nice separation and clean retention times keeping analyte a away from analyte b or analyte one away from analyte two and they're not just kind of all clustered together there okay but this can work against you when you're looking within the same band of analyte so let's say analyte two is going over these porous surface areas well they're not completely uniform and so certain analyte particles may get stuck or go down into the pores further than some of the others and so this can create a situation where some of your analyte in a given band may end up moving generally over the top of the surfaces of these packing materials and they're not really going to have much interaction within the pore and then you're going to have some that can go part way into the porous material right and then you can have some that are going to go even further deeper into the porous material and so a lot of the images that you'll see that kind of set this up it almost looks like a pac-man image okay but you've got sort of this bead here that's got the pores in it all right and then these are the modifications if you look at these little uh hair like structures right these are your c18s or whatever coming off here and so what you want to kind of keep in mind and let's bring this actually all the way up here okay that'll work better for the example what you're going to see here is that if your analyte is sort of coming across as a band here right and it's starting to move over top of this it is possible that some of the analyte is going to kind of take this pathway here and it's not really going to go into that pore but it's possible that there's going to be another version of that analyte let's pick something different than blue here so we can see it we'll do the purple and that's going to come across and that could potentially go down inside of here exposing it to more surface area and giving it a longer run time over that particular stationary phase particle okay and so this is the general concept of mass transfer is um as we're jumping particle to particle or we're literally taking the mass the analytes and moving them along the column um how long are they spending and how often are they transferring back and forth between the particles well that can be a factor here we need to consider the fact that most of these particles the stationary phase particles are porous and so the analytes may have a different amount of time that they're spending in each of these various pores there okay and so to wrap up as we're kind of talking about this before we get to minimizing it what you can see here is the flow rate it now multiplied with the mass transfer factor and that is because if you take a very short amount of time right this number is going to be low so that is to say if this value right here your flow rate is low let's say one milliliter per minute right if it's moving slow enough then the movement of the red analyte to the purple analyte really isn't going to make a huge amount of difference because the red analyte's not being pushed through that fast in comparison okay but as we start to rapidly increase and u starts to climb meaning our flow rate is increasing we keep bumping it up well now the rate of the one that is not interacting with those pores may start to separate out quite a bit ahead of the ones that are taking that extra porous trip through the packed material and again you can see how flow rate is playing in here there's probably a balance between factors b and c here in the uh equation right so when we look at the van temer equation it's really a mix of all of these that are going to be playing a factor here in regards to band broadening all right so what we want to address now that we kind of can visualize and conceptualize what each of these are let's talk about for each of these how we can limit or minimize the effects that are being played here and you're going to see some common themes so let's go ahead and start with that so how do we minimize band broadening here's our list that i just brought up now please keep in mind these are general suggestions so i cannot give you a specific column length or a specific flow rate here because some of that is going to depend on the particular solution that you are trying to separate and what those results look like okay but they are general principles to keep in mind when you are working with hplc so number one or letter a in the equation how do we handle multi-path diffusion and minimizing that well a lot of that is going to be on the producer of the stationary phase on their end so when you are purchasing your columns it is very important that you are going through a reputable company and one that you have a good relationship with and you can trust in terms of their processing so number one you need well packed columns because if the columns are not well packed and the stationary is not excuse me the stationary phase is not well packed that's going to leave larger openings between the particles which is going to allow for more pathways that the analyte can travel which could potentially start separating out and causing uh multi-path diffusion okay smaller stationary particles are better that is more within your control many times when you select a column you can pick the general particle size that you are going to have shipped to you and so generally speaking going with smaller stationary particles is a better idea and you're going to see that theme repeated here in a couple minutes all right so if you're confused about some of that i would encourage you to go back to the lecture that i gave on the chemistry behind columns which i want to say is maybe lecture two but go back through the playlist and check and there's a discussion about uh you know stationary phase size what's relatively considered acceptable when you could run into pressure issues things like that okay and then the other thing you want to make sure is you definitely want uniform size and distribution of the particles that are making up the stationary phase so again you're kind of at the uh mercy of the column producer okay but obviously taking good care of your columns uh plays into this in terms of making sure they continue to stay well packed and that the distribution is good okay so for the longitudinal diffusion this is our random or natural diffusion process okay moderate flow rates are best and i i make a note here careful not too high and not too low now both of these are important because if it is too low or too slow okay broadening can occur and we already kind of talked about that earlier but you need to be careful that it's not too fast either because if it is too fast and you're trying to run away from the broadening getting too fast with the flow rate can lead to back pressure issues and high pressure issues as you're trying to kind of force this mobile phase through the column faster than it should be going so you do potentially have pressure problems if you're trying to go too high with the flow rate okay so it's not great to say hey i'm going to minimize all my longitudinal diffusion as best i can but at the cost of creating pressure issues in the instrumentation okay that's just as problematic for different reasons so when you get ready to use any sort of tubing and the column is kind of beneath this it's the same premise you want to use the minimal amount that's required okay especially if analyte is passing through or going somewhere you want to make sure all your fixtures are correct that you're connecting using the right fixtures but you don't want excessive amounts of column length or tubing and you want to try to keep the diameter reasonable uh reasonably small okay because the larger it becomes the easier the longitudinal diffusion is going to take hold and in terms of column length that is a double edged sword because the shorter the column length the less the longitudinal diffusion but the longer the column length the better the overall general separation is going to be right because there's more uh theoretical plates there's more chance for that separation to occur but it you have to be careful because that longitudinal diffusion that broadening effect can come in so reasonable is kind of a subjective term here you need it to be reasonably long enough to separate out what your goal is or what kind of mixtures you're working with but not so long that you're promoting broadening or unneeded time on the column okay mass transfer so for the last factor smaller stationary phase particles are ideal again we're reinforcing this concept because the smaller the particles are the less the porous effects are going to be felt and the less the gap in mass transfer is going to be between analytes that are not interacting with those pores versus those that are heating the column can help with this okay so heating and again you need to be careful because heating could potentially uh increase the longitudinal diffusion so there's a balance but some heating may be a good experimental technique to use and do some trial and error that can generally help keep the non-porous and the porous particles a little more uniform in nature as we're moving them along okay and then again i put lower to moderate flow rates here instead of just moderate if you can stay on the lower end of moderate and still not promote too much longitudinal diffusion that's a good idea because the higher these rates go the more the mass transfer effect is going to play into this all right so that's it i'm going to wrap it up there that pretty much discusses what i wanted to talk about for band broadenings what is it why is it a problem what plays into it as well as how can we attempt to minimize it and there's some practical solutions here that you'd have to play back and forth with when you're working with your hplc factors okay so that is it for me again one more shout out to the website you can find it down in the link description chemcomplete.com go over there and pick yourself up a guide if you want to help support the channel and you want to inform yourself other than that remember to subscribe like if the content helped you comment and i'll get back to you and thank you so much for spending the time to learn with me i'm out of here until the next one guys take care