Introduction to HPLC - Lecture 1: HPLC Basics

hello everybody welcome to another chem complete lecture series and today's lecture series we are going to be introducing an introduction course to HPLC chromatography so HPLC stands for high performance liquid chromatography and in this first lecture we're going to take an overview of HPLC and learn some of the basics and then we will get into more detailed parts as the lecture series continues into different types of columns and how to manipulate mobile phases and things of that nature so let's go ahead and get started with a general overview the first thing that we want to

have an understanding of is what chromatography is so when we use the term chromatography we're talking about a separation of compounds now traditionally when chromatography was first discovered it was through basically just a separation on silica or alumina type gel and you could literally see the colors separating out from these different organic compounds and that's where the name chromatography comes from the chroma part with the color is referring to that separation where you could visually see colors now in today's more modern labs we usually have two options when it comes to instrumentation the first one

is gas chromatographs which is GC gas chromatography and then the second one which we will be talking about is obviously HPLC now the difference between these is when you want to use GC certainly you're going to be dealing with things going into a gas phase but one of the distinctions is that you need whatever you are putting through the GC to be volatile so whatever compounds are going to work with they have to be volatile compounds and that means that GC tends to be better situated for compounds that have a lower molecular weight and that's

simply because due to the volatility compounds with lower molecular weight have fewer intermolecular forces and therefore they tend to be more volatile and then obviously GC is going to be operating at higher temperatures because whatever volatile compounds we put in there we are going to need to push them into the gas phase and that requires a higher temperature both internally in the column and at the injection port where we are injecting our samples so on the other hand if we take a look at HPLC HPLC is acceptable for both volatile and you can do non-volatile

compounds in HPLC because we're not trying to push things into the gas phase it's liquid chromatography the only thing you really need to worry about is solubility now solubility is going to be important especially when we start talking about mobile phases and picking out solvents how things adhere to columns and how they are washed off of the columns and how that affects retention time okay and one of the other benefits of this is that it's less destructive than gas chromatography is you can recover your samples most of the time if it's you only have a

small amount and you're attempting to purify something by HPLC that is possible and one of the reasons it's also less destructive is most of the time you can run these at room temperature so around 25 degrees Celsius there are no high temperature requirements where you have to move these compounds into a gas phase so that's sort of an overview of chromatography the different methods we have available and which one might be useful in which case so the next thing that we want to ask is how does HPLC actually work so now that we know some

of the parameters we want to take a look at how HPLC actually separates compounds and works and the way that this occurs is there are two phases that we consider and this is usually true of any type of benchtop chrome photography as well so when we're talking about column chromatography the original chromatography we mentioned this is true so you're going to have a mobile phase and as the name implies this is going to be a phase involved in the movement of compounds through the column and your mobile phase in HPLC is going to be a

liquid and usually it's some sort of solvent or a mixture of water and organic solvents and you can manipulate the polarity of the mobile phase by mixing and matching so certainly water alcohols those are going to lead to more polar mobile systems and that will mobilize polar compounds whereas you can scale it back you could go mid range with something like ethyl acetate or methylene chloride and then you could scale back even further and if you really want to get more into the nonpolar region you could start working with maybe some hexanes or some of

your ethers where you would have a better movement of your nonpolar compounds in a situation like that alright so you have the mobile phase and then the other phase that you have is known as the stationary phase now the stationary phase again by the name is going to be a phase that is not involved with movement it is going to be keeping compounds sort of adhered or stuck where they are so this is usually going to be a solid chemical phase and this is the material that you're going to find in your column so it's

solid chemicals that are going to be bound into a column now you may have heard if you've ever experienced HPLC or GC because we use columns and GC as well you may have heard of the term column the column itself is just a very long tube and then the columns properties depend on what has been coated inside of the column so maybe you've heard of things and will show an example later something like a c18 column a c8 column see four column an ion exchange column all of those types of columns are going to have

a different interior makeup or a different interior stationery phase and that will be responsible for binding the compounds as they're moving through and you really have sort of a give-and-take between the mobile phase and the stationary phase so when you're doing this you will take you our sample it will be loaded with the auto-injector into the column along with some mobile phase and then your sample will adhere to the stationary phase so it almost like glue or some sort of a sticky pad the compounds will adhere to the stationary phase and then they will slowly

be washed along depending on their preference or their binding to the stationary phase so the mobile phase is pumped through at a steady rate and then depending on the polarity of that mobile phase and the polarity of the stationary phase that will determine how quickly your materials move along the column and the goal with HPLC is you want to separate these materials out as best you can you don't want them clustering together again going back to something like a column that you would be running on a bench top in a organic synthesis type of manner

when you have a column like that the goal is to separate the compounds out into different sort of Alcott's of solvent you don't want to just bleed all of them down together otherwise you haven't really done any type of chromatography and separation all right so to do a very simple picture and we will have a larger visual as we wrap this first lecture portion up in a little bit but to give a very simple diagram here the way that this would work is you have your mobile phase now I'm just going to draw a single

container of solvent okay many times in HPLC you're going to end up having several different solvents and the HPLC pumps can mix and match them at different rates so let's say it's called a solvent gradient I want to start with 50 percent methanol 50 percent water and then I want to move to 70 five percent methanol five minutes into the run for better separation at that point you can tell or program your HPLC to pick and choose different amounts that it puts through the pump at a given time all right but in general you're gonna

have your bottles filled with mobile phase and the mobile phase is going to be connected okay into the HPLC it'll go through the pumps it also goes through something called a de gasser which we will talk about a couple of minutes but the idea is that eventually okay along with your compound that is injected it's going to find its way onto the stationary phase which is your column okay and in the stationary phase it's going to as we described travel along and separate out at different times and once it comes off of the column it's

going to hit the detector so the detector is going to be using usually we use a lot of UV vis you can use a fluorometer e type of detector whether you're doing fluorescence if you have the proper derivatives and materials but you have to have some way of detecting what's coming off of that stationary phase and then that is what will be output into a chromatogram so that's where we see the actual graph and you've got the peaks as they go along and those Peaks have different tension times okay and again we'll talk about all

of this in a little bit more detail but the key here is that the compounds are going to travel at different rates and therefore they hit the detector at different time periods and those different times refer to as retention times is how we can really identify what is coming off of the stationary phase or the column all right so a mobile phase is oftentimes going to be a mixture of water and organic solvents when we take a look at it and the polarity of the mixture or the lack thereof because you could certainly be working

with nonpolar ones as well that will determine how long the compounds are going to stay on the column or adhere to the column and that's known as retention time and then the stationary phase the columns are packed with different materials usually some sort of organic materials often applied to a silica bed and those will bind to the compounds that are being separated and sort of spread them out as the mobile phase is going through doing its thing all right so one of the things I mentioned here was there are different types of material that get

packed in your columns and as the chemists you have to decide what type of column you want to pick out in order to get the best separation and each of those can go into their own lecture so we can talk about and we will make a list but we can talk about things such as ion exchange or reverse phase or normal phase hplc those all have different meanings they have different type of mobile phase and stationary phase setups right but for example if we were to talk about using AC 18 column AC 18 column I'm

drawing the inside of the column here okay so imagine that this is the inside of the column what you would find is a silica binding portion that sort of lines the bed of the column okay now a lot of times when we do wet bench sort of a desktop chromatography we use silica gel right or alumina so you have this as the base and then once you have your silica gel you can adhere other types of organic material to the side so what you end up with is in a c18 column you get very long

18 membered carbon chains so what I'm gonna write here is CH 2 we have 17 of those and then the 18th is going to be a ch3 right and then you would see that at every single silica site all right so it's gonna get a little crowded here but I'll draw another one all right so you'd go like this you have the silica with the two methyl groups and then you would have a ch2 right and you have 17 of those and a methyl so you have these very very long hydrophobic and that's important in

a case like this okay hydrophobic chains and I mentioned that that's important because you could also have a hydrophilic or a polar type of chain this one in particular is going to have better adherence to other nonpolar compounds alright and it would sort of go on where throughout the column you would have this type of a binding site and as the materials come through they interact through intermolecular forces with these other compounds here right these long chains that are hydrophobic and that sort of adheres your organic materials to the column and the mobile phase will

sort of push them along and depending on how strongly they adhere to it that is going to be what determines how quickly they will come off of a column which is relating to retention time there so for something like a c18 you're dealing with a highly nonpolar column so nonpolar analytes are going to be retained at a longer rate right they're gonna stay on the column longer before they get pushed off onto the detector hey now this is very important the choice of column and solvent phases will always play a role in your separations and

you want to basically maximize the efficiency of those separations which again could be its own lecture in terms of how do we maximize retention time how do we get good resolution so the peaks aren't too clustered together and we'll address that as we go along in this course so what are some of the modes of HPLC that we can talk about because if you remember I mentioned that there are several modes and each one of them could have their own lecture in terms of what's involved so the first mode that we would talk about is

just normal okay and for a normal phase you would usually be talking about a situation where the column is more taller than the mobile phase so the mobile phase is going to be nonpolar in that case and the column is going to be more polar hey one of the other very common ones is reverse phase a CA teens tend to be used in Reverse phase a lot and with a reverse phase hplc it's just the opposite of what we said so it's something like a c18 you have a very nonpolar column but then your mobile

phase tends to be more polar so you might have methanol see the nitrile water things of that nature in different ratios okay then you've got some other ones when you start dealing with ion pair okay so you can have an ion pair and you can also have an ion exchange phase and then one of the last ones that sometimes gets mentioned is you can also have a size exclusion chromatography okay so each of these has their own unique way of dealing with stuff so sometimes when you're dealing with the ion exchange columns you might be

using some sort of a buffer like a sodium buffer at different concentrations whereas when you're doing just the regular phase or the reverse phase you are using water and organic solvents instead of a salt or a buffer system alright so each of these is its own lecture as I mentioned but I did want to list these here so that we could sort of talk about them briefly and give you an idea of some of the upcoming content we will cover each of these in its own right but it'll be its own probably you know 20

minute 30 minute lecture dealing with everything that goes into each one of these types of phases so with that being said what I want to do I want to bring up an image here as we get ready to start wrapping up this first lecture and really go through each of the parts of an HPLC because it's one thing to talk about it in theory with this sort of like simple drawing I have up here in blue but it's another thing when you actually walk into a lab and you see the HPLC there's a lot of

containers or boxes for this instrument that are sort of stacked on top of one another so I made a rough sketch of an HPLC setup it's very generalized but it'll get the point across as we're talking about this so let me bring that up and we'll take a look at that together okay so here we are we have the setup for what the HPLC would ideally look like if we had the system and we could kind of briefly look at the insides again this is still a simplified drawing but it's more detailed than what I

showed a few minutes ago so the very top part here you can see the bottles that are hanging out at the top this is where you're going to find your mobile phase components okay so these are your solvents your water whatever it might be it could be your buffer solutions but your mobile phase is usually found at the top of the HPLC instrument and it will be in you know maybe four liter solvent bottles it might be in a one liter plastic container again most the time you're gonna see multiple containers because many HPLC techniques

use what's called a gradient solvent system where you're changing it over time and either increasing or decreasing the polarity at certain time markings on the column so that's what's being hosted at the top there where you see the different colored bottles those are your mobile phases so the mobile phases send the liquids into the next component and that's the component we see right here and that is known as ad gasser now ad gasser has one simple job and that is literally two D gas the solution so the de gasser as it accepts the mobile phase

these solvents it's attempting to remove any air bubbles or any sort of air pockets that might be found in your mobile phase because you cannot have where you certainly don't want to have air pockets as you're going along in your column because that can disrupt the flow and the rate at which these components come out it can screw up your base fine particularly so having a good degassing system is crucial in making sure that you establish a steady baseline and you have nice clean separation of Peaks when they finally hit the detector and you get

your chromatogram so the mobile phase is pumped into the de gasser and after any type of air is removed this liquid comes down and is sent to the pump system so these are the pumps now when you have your HPLC pumps they use a back pressure in order to continuously and it is very important that it's continuous and at a steady rate move the mobile phase down through the rest of the HPLC system across the column and into the detector and it's important that your pumps are in good shape in order to maintain this regular

flow okay which you could call the flow rate of solvent you can adjust how quickly these pumps are working and at what pressure in order to change the flow rate when you change the flow rate you obviously change how quickly or slowly things will come off of your column and hit the detector so changing any of your pump settings will be able to change the retention times for the compounds so sometimes that can be a good thing sometimes that can be a bad thing all right well you've got your pumps they are going to help

mix these mobile phases and then pump them down and get them ready to go into the stationary phase all right so after the pumps have thoroughly mixed it it's going to come in to the next part of the HPLC in which we have the mixing for the autosampler and the injector alright so what you see on the left over here is your auto sampler or at least my best representation of one okay and the auto sampler is going to take your samples so these little blue vials here okay are your samples you've processed them in

a lab you've been you know given these samples you put them into a rack you load that into the auto sampler and then through the software the autosampler will come pick up give an amount you can choose the injection amount it's usually a tiny amount like five not five hundred fifty to a hundred microliters excuse me and that will send this material and mix it right into the injection port where it will also meet up with the mobile phase and that will be sent down into the column so that's the next part right here right

after everything is thoroughly mixed it is sent down and your material from the autoloader is going to adhere to the column and then your pump is doing its thing it's continuously pushing the mobile phase along so your materials will start traveling on the inside of your column at a given rate right and we've talked about the inside of the column and how that works the adherence and then at some point they will come off of the column they will be sent down into the detector so again your detector could be fluorescence it could be UV

those are probably the two most common but when you hit the detector that is where the signals are going to be received and then you can output into a chromatogram and when you get to the chromatogram that's the actual printout that you would be looking at where you have the peaks and their retention times and that is the final step for the HPLC at that point you would be using software in order to analyze your actual chromatogram okay so a general chromatogram is going to look something like this you're going to have so along the

y-axis you're going to usually some see some sort of a response okay well usually it's just called abundance or detector response or something of that nature and then on the x-axis you're going to see the time this is typically in minutes it could be in a different unit depending on how you set it up but it's almost always seen in minutes and so you have your baseline again to have a steady baseline you need to make sure that you've got a good degassed sir for your mobile phase and then at some point you're gonna see

if you have compound peaks that start coming off and now you might just have one peak if it's a pure substance but most of the time when you're doing HPLC it's to separate out several components from one another right so you can see an example like this now there's two important parts here and that is you have what's called T naught or T 0 which is the initial amount of time that it takes for anything that has not adhered to the column to just come out and directly be flushed okay so there is a compound

called uracil that is typically utilized in order to detect when this hits however again T naught is going to be the aleutian time for anything that was not retained so for on retained material and when you have that sometimes you'll hear it called or referred to as dead time again this is simply time that elapses when you have essentially the first dump of mobile phase into the detector that did not have any type of adherence to the call all right so the other important thing that we've been talking about over and over again is the

retention time now this goes certainly beyond the dead time that we have there out to whatever peak you're currently looking at so let's say that we're looking at this last peak right here the amount of time that it takes to go from the dead time out here is TR and that is considered to be the time of retention all right now when we start talking about things like resolution in the distance between Peaks it's going to be important to understand both of these because both of these components are used in calculating how to improve things

like resolution okay but retention time hey and what's important about retention time and I know I mentioned this once earlier is that this is really we can identify compounds by retention time so the compound identity is associated by retention time and that's really how we look at compounds when we do HPLC now one of the other things that we discuss with HPLC is we do discuss the area under the peak so the retention time kind of identifies what compound we're working with the area under the peak is going to tell us how much of that

compound there is ok so there's essentially the area under the peak is going to be proportional to the analyte amount now the way that you determine this because you have to have something to compare it to is that you would need to use a calibration curve so let's say that I'm working with amino acids and I want to analyze the amount of lysine so if I'm going to analyze the amount of lysine I need to have a calibration curve of lysine at several different concentrations and then ideally I want that range to cover from min

to max any type of sample of lysine that I might be expecting so we can be a little bit of trial and error when you're first setting up your calibration curve if you don't know a whole lot about your samples but once you have a reliable one you'll know the area under the peak for the various concentrations of your lysine and therefore you would be able to determine the amount based on any sort of unknown sample going through and you always know that it's lysine based on the retention time so that would just be a

example there all right so we're closing in on half an hour I think I'm going to but up there in terms of the introduction to HPLC and we will continue forward with these lessons and start to outline some more detailed parts to the HPLC particularly the different types of phases that we can undergo as well as some troubleshooting stuff so what happens if all my Peaks are too close together how can I improve that things of that nature so hopefully everybody found this introduction course to be useful I hope that we will see you for

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