Transcript for:
Normal Phase HPLC Overview

hi there everyone welcome to another chem complete lecture i am professor tomney and let me just say this lecture is a long overdue one and it has been requested by many people over the past months so today we are finally going to continue the hplc series and we're going to learn about the next hplc phase typing which believe it or not is known as normal phase and with such an uninspired name and it's very basic sounding terminology you would think that we should have covered this at the start of the hplc lectures but some of our prior lectures on things like uh reverse phase and ion-based hplc came first because they are actually more common than normal phase hplc is [Music] okay so before we get into this remember that info for anything i'm discussing here will be referenced down in the description box including a link to my personal website at chemcomplete.com you can always go there to check me out and the hplc playlist so some of the terminology we might be using here may be a little confusing if you're just jumping into normal phase and you have don't have some of the back reference especially the first two lectures which was kind of the intro to hplc as well as the hplc uh basics for columns and understanding columns how they work and some of the features with that okay so as always i appreciate all the love and support that you guys have shown um and i do have some exciting news i'm going to be finished with my introduction to hplc guide later this month so if you are viewing this video in late august of 2021 or at some time beyond that there should be a link down in the description for the guide and you can still always go over and look at the previous guides that i have produced at chemcomplete.com and that's an excellent way to support my work and remember that always just liking any useful content you come across and engaging with the channel is support enough already and i love all the support that you guys have given me we're well over 10 000 subscribers now so welcome back to the channel and let's take a look at this so as far as normal phase hplc we're going to discuss a couple of things so what is normal phase hplc and why do we actually call it normal phase it's a bit bizarre right and we've got the normal phase columns and solvents we want to have a discussion about that how are they different from some of the other things that we've come across so far how are they similar something called localization action this is a very important concept in chemistry in general but it is especially important when you start talking about adherence to stationary phases and columns so we'll talk about that and then we obviously want to know when should we be applying normal phase chromatography um in comparison to some of the other methods and then finally we usually like to wrap up with some kind of pros and cons or advantages and drawbacks to using normal phase chromatography so we're going to take a look at each of these individually all right so first of all what is normal phase hplc and why do we call it that well the reason that it's called normal phase hplc is that it was originally one of the it was the first way that hplc was utilized or implemented okay so if you follow this lecture series so far you may remember that reverse phase hplc was one of the first methods that we really talked about after column basics and stuff like that and so the name here when it says normal phase hplc this setup was originally the hplc conditions used and developed prior to the development of reverse phase hplc so that is the reasoning behind the quote-unquote normal name okay so it's usually uh considered a secondary option to reverse phase so and we'll talk about that more soon reverse phase tends to be the one that is prioritized for research and development even though normal phase came first but that's why it's called normal is it was the first one present and then reverse phase was developed sort of with the reverse conditions uh compared to the normal phase hplc so understanding that right normal phase hplc is going to utilize a polar stationary phase or a polar column okay so if you remember in the discussions revolving around the reverse phase hplc we had the opposite set up with a non-polar column so we were talking about things like c18 columns with these long hydrocarbon chunks that would sort of be modified onto a silica base whereas now we are talking about a polar column instead of a non-polar column right so this is going to be a polar column which again we're talking about the stationary phase of the separatory process is polar in normal hplc okay now in counter to that our mobile phase is going to mostly be comprised of nonpolar solvents right so this is the liquid that we will be moving through to mobilize the analytes through the column and these are going to be relatively nonpolar whereas again in the reverse phase which is the more common technique utilized today and more prevalent in the literature the reverse phase used primarily polar solvents with water kind of topping that list water generally speaking is a big no-no in the normal phase hplc especially if you're dealing with something like a bare silica column because then if you don't have those modifications the water can kind of almost act as a modification and we'll talk about it that in the the pros and cons section uh but you do have to be kind of more aware if you're going to have water present and obviously water is present in our everyday environments we're talking about residual moisture and things like that you have to be aware of that when you're using normal hplc conditions where it might not be quite as critical a uh concern or consideration when you're dealing with reverse phase hplc okay so let's break this down further so column wise we said you're going to be sticking to polar options so one of the best known options okay for the columns and let's just label that here we'll say this is columns so one of the best options that we have for columns is going to be the bare silica or sometimes this is called non-modified silica based column right so just your classic silica column without any type of moderations or i'm sorry modifications i should say so this is similar to actually many large scale bench top columns that are used for large-scale purification in laboratory practices so i remember back when i was in graduate school i was doing organic chemistry right you would produce your synthesis or you would create your new compound and then there would usually be side products or leftover starting material if it wasn't completely efficient in conversion or whatever and you had to separate the materials out to purify your bulk product to take it to the next step in the synthetic process and one of the things we would utilize was silica columns it was very very effective at kind of on a larger scale larger than hplc separating out grams of material right hundreds of milligrams to grams of material at a time could be used with this bench top type of process and most of the solvent options were primarily non-polar so you know you're talking about your hexanes and your ethyl acetate mixtures maybe threw in a little tiny bit of methanol a couple drops or something to help with the polarity uh but generally speaking it was a non-polar approach from the mobile phase so coming back to this not just silica but there are other options that we could use for modifying a silica base okay so some of the functional groups that you might come across for this could also include things like cyano columns okay so this would be when you have some sort of a cyano which is also sometimes called a nitrile okay modification onto a silica column and we did talk about or see some of this sort of stuff towards the bottom edge of the reverse phase right so with the reverse face it was kind of like all right c18 c8 c4 and then if that still wasn't working maybe you could start kind of dabbling in the cyano adaptations right so if you wanted to get a little more polar than that you could get something like a diol modification so die meaning two and all meaning alcohols so this would be some sort of a column that the silica base would have an adaptation that has two alcohol groups right attached to it that's obviously extremely polar you think of the structure of water and how polar water is that's going to give you a lot of polarity on that mobile phase and then we can also deal with amino groups so you could consider right some sort of a nh or some sort of an nr where r is probably a small hydrocarbon not very large otherwise we're starting to talk nonpolar again and those would be some of the base modifications that you could do to these types of columns and you would get varying degrees of polarity and therefore varying degrees of retention and interactivity with the analytes that are coming across right so then let's talk about solvents so for normal hplc the solvents are primarily nonpolar as we've discussed a moment ago so the most common uh certainly that i've come across is usually hexane now there's a couple others that you could potentially use some people do in special cases use fluorinated solvents but hexane tends to be a more common choice at least from what i've seen and hexane is usually going to be kind of the primary uh solvent that you would be using it would make up the bulk of what you're using most of the time and that's because it is very nonpolar it's just a strict hydrocarbon without any type of polar functional groups or modifications there okay and then you're going to have other options that you can kind of utilize and couple with your base or your primary solvent in order to offer like sort of small bumps in polarity so an example of this would be ethyl acetate which i mentioned a minute ago okay ethyl acetate is a very common solvent that's used in conjunction with hexane and you kind of mix the ratios together try different percentages a lot of this will be trial and error but then you see what you can come across and what you can find that sort of optimizes the retention and the separation for what you're interested in okay another example is methylene chloride sometimes referred to as dichloromethane depending on the name that you're using okay and chloroform could be kind of ranked in here with this but some of the chlorinated solvents uh offer good choices you have to be careful when you're handling them really all of these but the chlorinated ones tend to have toxicity or carcinogenic concerns associated with them right so this for example would be uh ch2cl2 chloroform would be with three chlorines and one hydrogen there but those do offer kind of an optimal chlorinated solvent that's not too polar but does offer a little bit of a polarity bump if you're mixing it in with some of the others okay um some of your other ones now you have to start being a little more careful and selective when you're picking these out because we're starting to get more and more polar uh ethers and acetonitrile now if you remember and you go back to the um lecture on the reverse phase acetonitrile and ethers were actually at the bottom of that solvent list and that solvent list was the reverse of this so it said okay water is one of the go-to's and that's usually your primary one and then you have your alcohols methanol ethanol things like that right then you have acetonitrile and then you have ethers and ethers were kind of like okay this is middle of the world road polarity and that's generally where we stopped in that reverse phase lecture well now we're coming here and we're saying okay start very nonpolar hexane ethyl acetate right and then we cap out this is considered the more polar end right mid-level polarity for the normal phase so you're kind of seeing uh right a meeting in the middle where ethers and acetonitrile can sometimes cross over into both types of hplc techniques and then on the cusp or the outer edges are those extremely nonpolar or polar solvents that are usually reserved and that's not to say you might not have a small bit of methanol or something like that thrown in in some of your normal phase but generally speaking you're sticking more towards these uh that are going to make up the primary targets for your solvents in a mobile phase in normal uh phase hplc okay so with these options nailed down we now want to ask ourselves how does normal phase hplc work on sort of a mechanistic or a practical level so starting with the loading and separating of multiple analytes you can expect that analytes with polar functional groups are going to have strong interactions with the column or the stationary phase and that's because that column or stationary phase is also polar so this is primarily going to be due to intense inter-molecular forces so we're going to this is a very common concept out of general chemistry we're going to abbreviate imf right but if you imagine that you've got sort of this uh silica based uh level right and then let's just say we'll go with a diol for instance right so you've got this diol modification that's coming off of here when you have other polar components so let's say something that's got a hydroxyl group right coming across here you are going to have a very strong interaction between the partial positive hydroxyl uh and the partial negative associated with the dials and you're going to almost like a intermolecular glue you're going to get an interaction here with the column and the column's going to want to hold on to this analyte and keep it there right those forces are very attractive and so you the job of the mobile phase as the mobile phase is coming along here it's trying to slowly push right these interactions along the column until we can finally elute off the other side and you start collecting and the elution rates or the retention times are different for each of these compounds based on their polarity and how strongly or weakly they are going to interact with the stationary phase in the column so this is the uh technique or i shouldn't say technique but the phenomenon of localization so we were talking about in the beginning localization action and this is uh sort of that being conceptualized on an image level okay and it is important to conceptualize this because this is essentially how you are going to be separating your analytes in the column so the more polar analytes are going to adhere to the column longer and they'll have higher retention times this allows you to plan and predict how things are going to kind of separate out if you're setting up a normal phase situation okay so again this phenomenon of intermolecular adherence to the column is often referred to as the process of localization okay in which analytes are going to localize or stay in one area of the column and then we'll kind of be separated through the illusion of nonpolar solvents maybe with some slight increasing polarity if you try to do like a phase gradient sort of thing okay you have to if you're going to kind of do like phase gradient uh hplc you need to make sure that your solvents won't demix that they will stay mixed thoroughly uh when you're doing that sometimes that can be a larger concern when you're on the non-polar end okay but the nonpolar analytes we would expect them to come off of the column first and then the more polar analytes with that localization or adherence are going to be coming off the column last as far as retention time is concerned okay so when a non-polar mobile phase is used it easily moves the other nonpolar analytes off of the column giving them earlier retention times and then oftentimes slightly stronger polar solvents may be needed to kind of slowly break apart the localization and usher it along in the process so many times several different solvent ratios or combinations will be required for a little bit of a trial and error discovery process like i stated earlier research and development whatever you want to call it right in order to optimize the separation and the resolution of the hplc results because this is what you're working with here you need to move these along in a fashion right breaking apart the localization at a sufficient enough rate that you're not going through boat loads of solvent it doesn't take 20 you know plus hours just to do a small load or a small run but you also need to consider you don't want to wash everything off of the column all at once and not get any separation at all by adding something to polar like you know spiking it with 50 methanol or something like that that's far more likely to be an appropriate mixture when you're talking about reverse phase hplc instead of normal phase okay so this is kind of the action the mechanism of action and how this works so knowing this it still stands that reverse phase and many of the ion phases that we mentioned sort of at the beginning of this lecture are used over normal phase hplc they are preferred methods as far as uh trying to deal with these sort of separations right when research and development needs to turn to something usually reverse phase is the favored and if there's something very polar then maybe ion uh separation is utilized in some form okay so when is it appropriate to use normal phase chromatography well it turns out that there could be several scenarios uh in which this technique is applicable and that's what we want to take a look at or discuss next here all right so let's put sort of when to use normal phase which i'll abbreviate np normal phase hplc right so when is this appropriate well there are a limited number of cases but there are some so number one if you are going to be dealing with large bunches of hot primarily hydrocarbon based and i say primarily because obviously if there are certain uh functional groups that are present that increase the polarity that could change the situation okay but uh some examples right of something like this could be if you take a look at a lot of the vitamins right so maybe something like vitamin d which is kind of cholesterol-based cholesterol is this big bulky hydrocarbon steric molecule doesn't have a whole lot of polarity associated with it that's why it's solubilized in a lot of fats when we talk about it nutritionally okay vitamin a if you take a look at retinol or beta-carotene which is kind of the prelude to uh vitamin a it is very very long conjugated hydrocarbon molecule it's kind of this straight big chain okay so vitamins and again if maybe you're taking a look at something like hormones um basically if something is cholesterol based so it doesn't necessarily mean that it's cholesterol itself although that is important but if it is cholesterol based which a lot of hormones tend to be then it may be appropriate to use something like this but the key is if you've got large hydrocarbon molecules that primarily have a lot of c h groupings okay you're kind of steering away from a lot of the oxygens the nitrogens especially if those groupings have hydrogens on them so hydroxyl groups or alcohols amines though now you're starting to get pretty polar right and so this will generally be a good method for separating these types of compounds and again having some polarity is not necessarily bad because it helps them with the localization so they separate at different rates you don't want everything to just wash off at once but if you start getting very very polar molecules then that can become problematic because the localization is going to become so strong that it's going to be difficult to get decent separation it's either it's going to be all or none they kind of glue to the column or you put something in that's so polar that it just washes them all off well it's not going to separate or offer you anything if it's just all sticking to the column and adhering there or if it's all just being washed out and you basically get what you started with as far as the sample that you put in or loaded onto the column right okay so very often these compounds have poor solubility in polar solvent so remember reverse phase uses polar solvents right and so it can be next to impossible to load these compounds and even if you're lucky enough to load them moving these large hydrophobic compounds along something like a c18 column might be incredibly difficult okay so another situation which may seem extremely obvious uh but is still something we want to mark here is if reverse phase hplc fails okay so uh if this is the case usually what's gonna happen this is just reality here for a minute most people are gonna go to reverse phase unless they have a very strong reason like in example one and i'll give you another one a third example here but most people are going to go to reverse phase to start with uh and there's multiple reasons for that it's not just that the column is non-polar and that the mobile phase is polar but it think about the cost base if you're using water as your primary solvent for an hplc system in comparison to hexane and this is getting a little bit of a preview into the pros and cons category obviously one of those is going to be far more affordable than the other right if i'm buying a lot of hydrocarbon specialty solvents in comparison to uh water or some of the mass-produced alcohols that tend to be a lot more affordable that is something to consider okay and you have to keep in mind that when you're doing this cost is a factor right so it's one thing to sit and talk about the theory oh this is how it works and this how you can separate and these are how you optimize but it's very different if you're let's say a medium-sized company and you are offering clients hplc runs on a consistent basis to just turn around and say oh well we'll just use tons and tons of hexane right now there's certain ways that you can kind of get around some of that you can try to recover some of it which is a pro because they have low boiling points but generally speaking you need to consider the amount of waste being generated the bottom line in terms of your dollars all of this can play into right something in relation to this so um what we have here right reverse phase hplc fails then normal phase may be a serious consideration after that okay and number three and this one's kind of interesting okay is that you can very often separate out geometrical isomers okay so this is kind of a large category but this is important because geometrical isomers can be very difficult to separate chemically because they have the same exact chemical components they have the same oxygens and hydrogens right so think of something so to give a kind of a sub-example here right think of something like enantiomers for those of you that have some background in stereochemistry okay so enantiomers are chemical molecules that are essentially going to be the non-superimposable mirror images of one another um right and we know that this is important in the drug world you have to be able to separate them out because they could have different binding receptors in the body but it's very difficult if they have the same exact chemical functional groups and sort of chemical identity to be able to take them and separate them out and say oh well i'll just solubilize this uh you know set of enantiomers in a water and ethyl acetate uh extraction because they're both going to go into the same phase they have the same general chemical properties uh when you're taking a look at them so that's having the ability to separate this out is very very important okay so to give you an example let's take a look at a column bed where there's two different enantiomers and one may be separated out uh compared to another maybe not enantiomers excuse me but geometrical isomer so give me a second and i will set that up okay so to illustrate this example let's consider a bare silica column this time all right so we've got sort of these silica based functionalities that have the hydroxyl coming off it right that's the bare silica and you can see this along with the dial columns these are going to interact strongly with water which again i'm kind of previewing uh water needs to be handled with care in these types of environments so you end up having a situation like this okay where you've got right so we've got the column this is the stationary phase going this way and if you take a look at this let's consider two different compounds so for one we are going to have a para chlorobenzene or dichloro right there okay so para is a 1 4 positioning like this okay and we're also going to consider its geometrical isomer which will be the meta version so we're going to do here's chlorine and here's chlorine right so this sort of has a 1 4 substitution pattern whereas this has a 1 3 substitution pattern for those of you that may be a little far away from organic chemistry at the moment and haven't heard that in a while okay but they are right the same number of hydrogens carbons chlorines present they're just arranged in a different fashion so you could see where this might become problematic when you say oh i'll just throw these into this type of extraction or i'll separate them out by boiling or things like that they are going to have very similar properties to one another but what's interesting here is that when you put them on the column kind of like when they go into a human body enantiomers or geometrical isomers have the ability to bond in certain geometrical fashions and therefore a lot of times they can be separated out if you have that selective binding capability so if we have a situation right where we've got something that comes along like this through the column that's a chlorine there right in the 1 4 well this is just perfectly aligned right if these are partially negative these are perfectly aligned to just have a nice interaction here right with the silica hydroxyl groups however all i have to do is change the chlorine to this position and now all of the sudden right i don't have that interaction anymore even if i change it here right is the alignment isn't going to be set up effectively there and so it turns out that you could effectively separate a 1 4 versus a 1 3 isomer when you have the ability to localize certain geometrical isomers over others on these columns and this is a huge advantage so while there may be limited scope to normal phase hplc the ability to separate geometrical isomer should not be understated or undersold because it generally speaking is very difficult to do you need to have the ability to selectively bind to certain geometrical arrangements over others again this is quite effective in living systems where certain enzymes can bind to or pick up one compound over another but generally speaking we don't want to be using expensive enzymes or cell lines or certainly humans right in order to go through and try to separate out and do these trials so uh hplc uh normal phase is effective at being able to do this and that is something again that should not be undersold in terms of the benefits of normal hplc so then finally what we're going to kind of do here is wrap up because we're going on about half an hour for this one and we're going to talk about some of the pros and the cons so now that we know the groundwork for normal hplc how it works at the molecular level and when it might be appropriate to use we're going to wrap up the lesson with a compare and contrast regarding the advantages and disadvantages so let's consider all of the advantages for a moment and then we will talk about some of the drawbacks that could come with that doing a pros and cons list so some of the pros for normal phase chromatography are going to be the following so this is our pro list here okay the first one is that there's going to be a larger range of and i'm going to abbreviate this here hydrocarbon okay so there's a larger range of hydrocarbon compounds that are going to be soluble in the solvents used so the mobile phase so this kind of goes back to the first point here right which is that if you have very large hydrocarbon compounds or any compounds that are primarily hydrocarbon in nature without a whole lot of excess polarity you are going to have difficulty using alcohols and water and some of the polar solvents used in reverse phase to solubilize them load them onto a column and move them through so this is definitely a pro or an advantage of the normal phase okay now one that may be overlooked sometimes is that because we're using these non-polar solvents the solvents that tend to be used here are going to have a lower viscosity now viscosity is a chemical term uh that is used to sort of describe the flow how thick a liquid is okay so we would say something is very viscous if it pours slow so for instance maple syrup okay whereas something is not as viscous if it pours very readily or rapidly like water but even within that if you start looking at hexane and you start looking at ethyl acetate a lot of the ethers the hexanes they are not as viscous as water and the alcohols and that's because they don't have the same intermolecular forces that kind of bind them together or glue them together and so when you get those weaker intermolecular forces the viscosity goes down meaning it's more free-flowing and it doesn't kind of plug up as easily okay so why is this a benefit well it's going to allow okay in turn higher or increased flow rates without a problem and why is that beneficial because you can move your system your hpl system quicker you can move samples through it at a more ready rate you can prep your columns and flush them at a quicker rate generally speaking time is money you save time when you're using less viscous solvents now that doesn't mean that you always can but in the case that normal phase works out this is a pro is that you're going to be able to kind of run your instrument at a higher flow rate and the higher the flow rate goes generally speaking the quicker the run is going to be okay so that's an important pro to keep in mind kind of a practical one because you're really talking about saving time in the long run if you're doing these techniques over and over again day in and day out all right okay now another thing to consider is that generally speaking there's going to be a wide variety of nonpolar solvents um you know there's still a decent variety of polar ones but generally speaking there are a lot of different ones that would be considered nonpolar up to mild polarity and so when you have a wide variety of solvents that are sort of at your disposal for mixing and matching in these situations you usually can have a high level of control or specificity over retention okay and that can be beneficial because if you can manipulate your solvents or you have a whole lot of them and you can kind of tinker with them and play around if you have a high level control over retention you can leave certain things on the column while removing others and getting a lot of distance between them to get very clean separation with your hplc so having a large kind of arsenal of non-polar organic solvents is going to allow for a high degree of retention time selectivity and separation and so that's a pro okay one of the others and i'm just going to kind of write this here this is still a recap of what we had above is that you can utilize this for geometric isomers okay they tend to be difficult to separate out and so having the ability to do this is really a pro or a positive in the normal phase system okay and then one of the last things that's important and this kind of balances itself out with one of the cons that we'll see in a minute is that the solvents generally speaking because they're nonpolar they're not as viscous they don't have as strong of an intermolecular force the solvents are going to have lower boiling points and if you have solvents that have lower boiling points they are usually going to be easier to recover and that's important because when you talk about green chemistry especially with these solvents that potentially are going to be damaging to the environment damaging to the wallet produce a lot of waste you want to be able to recover them with a low boiling point to utilize them over and over again okay so there are various devices for instance when i was in graduate school this was on the bench top level uh we would use things called rotovaps and they were basically round bottom flasks that would sit in a water bath and you would set it to a very low or mild temperature you'd pull a vacuum and then it would remove the solvent system you were utilizing away from the analyte or the compound or the product and then recondense it over a cold column with some antifreeze running through it right chilling it down some chilling solution and then you could recover your solvent and if you were using that solvent to run those columns on let's say a weekly basis a daily basis that solvent is very valuable to you you want to recover it and be able to utilize it again right instead of just throwing it into the waste obviously if you're doing something that needs to be excessively pure you might need to go through multiple rounds of this just to make sure that there's no analyte especially if you have something that's a lower boiling point analyte right that's making it in there but generally speaking you are able to recover some of these solvents which can help ease some of the burden on the pocketbook okay so now some of the cons to wrap this up and the first one is going to be kind of the opposite of the first up here so while there's a large range of hydrocarbon compounds that are soluble in the solvent normal phase is not good at dealing with polarity okay so it's not good with i what i should say is it's not good with high polarity okay so very polar functional groups uh ie ions right we're going to need to be sticking more to reverse phase or even a subset of reverse phase like the ion pairing chromatography okay so some of the reasons for this is that you've got high degrees of localization that are going to be difficult to break apart so we talked about that right local localization okay we're going to also have a poor ability to elute with the inability to use highly polar solvents so generally speaking it's going to be porous solvent illusion in a case like that okay so one of the other things you've heard me mentioning as we've gone out here is that water typically needs to be avoided this is particularly so i'm going to kind of put a star with this one this is particularly uh you need to be careful of this with the bare silica because it can kind of set up and almost make it its own um addition to the bare silica column with how strong the adherence can be okay so water generally speaking needs to be avoided and this can be uh difficult because to avoid water is you're talking about moisture in the air moisture that can get into your solvents especially as you're starting to increase the polarity of your solvents you need to make sure that they're kept very dry right because you can certainly start getting water into some of your alcohols into your see the nitrile there's no reason that that can't absorb some of the moisture that's sitting around in the lab over time so you need to be very careful to avoid water in some of these situations and one of the reasons for that is that when the water kind of randomly combined throughout the column this can start to produce unpredictable separation and unpredictable results and when we start talking about unpredictable results or in other words results that are not going to be repeatable that is a problem in science right we should be able to use the same solvent system utilize it over and over again with the same column and generally speaking provided the columns in good health and has not been worn down too much you should be able to reproduce your results as far as retention times it shouldn't be spotty or changing back and forth on a day-to-day basis okay and then finally one last con to wrap it up because we're now at 40 minutes here okay the solvent cost okay and we're going to put and environmental impact impact is high okay so if you take a look and i talk about this some in my guide in terms of the cost um if you take a look at like four liters of hexane in comparison to 4 liters of hplc grade water or methanol it is cost prohibitive uh to do the hexane in comparison right so you can do it uh you have to pass costs along to the powers that be or the client or whoever it might be but generally speaking your cost to the bottom line is going to be higher and the cost also due to having to get rid of the waste in a certain way is going to be higher because there's greater environmental impact on a bunch of hexane that is produced as waste in comparison to a bunch of water that is produced as waste okay so that is going to be it we're going to wrap up our lecture on normal phase hplc here i hope you guys found it useful and you know a little bit more about normal phase hplc now so if you found this content useful i will encourage you to share it with others and like the content as it will help spread awareness with the youtube algorithms on what i'm working to teach here and the bottom line is i'm here to teach you i want to help you the viewer the chemist the student the person engaging with my content okay so subscriptions and comments are always appreciated and again you can always show your support by buying one of our guides or visiting chemcomplete.com you can reach out to me i am professor tomney i have more work to put together for you guys because it's been quite some time so i will see you in the next one take care everybody have a good one