all right let's resume chromatography then so there are four different types of chromatography that we talk about um the first one is partition chromatography so in partition hold on okay in partition chromatography we have it's gonna work there you go separation utilizing partition of the solute between two liquid phases so a lot of times we'll think of this as um it's called different separations so liquid liquid separation where you actually have two liquid phases and you're just having a transfer between the two phases so we do have two different types of partition chromatography one is called liquid liquid chromatography and liquid liquid chromatography is in which you have a liquid stationary phase retained on the surface of the packing material or the packing by physical adsorption one common disadvantage to liquid liquid chromatography is is that you end up a lot of times with loss of the stationary phase by dissolution in the mobile phase the solution in the mobile phase all right so the second type of partition chromatography is going to be bonded phase chromatography and in this type the supports are prepared from a rigid silica composition the main difference between liquid liquid chromatography and bonded phase is the method by which the stationary phase is held on the support so in liquid liquid you have it's retained on the surface by an absorption and bonded phase it's going to be in the actual preparation of the composition of the stationary phase the second type of chromatography is absorption chromatography or liquid solid chromatography and in here you have a stationary phase and that's eve typically gonna either be silica or alumina is an adsorbent typically you will see that stationary phase of silicon or alumina because it has a high sample capacity and so a higher sample capacity allows for a greater sample to go through the column or through the separation process all right okay so the next type is ion exchange chromatography so unfortunately those of you that had me in analytical this last year we didn't have an opportunity to do the iron exchange chromatography but this is one of the most common um separation chromatographies we do it includes a resin so it's you utilize utilize an ion exchange resin and as the name implies you're going to exchange out ions so your resin is either going to be you can either have a cation or an anion resin and it's going to be loaded so for instance the one that we would in lab is you would actually run it was a cation exchange resin and you load it with excess protons excess hydrogens and we do that through using a concentrated or a higher concentrated acid then you always want to use something that has a lower affinity than your whatever you're trying to exchange and so in the example we didn't would have done lab was we exchanged calcium for the protons and then the calcium sticks to the resin the protons are released from the resin and you can figure out how much was exchanged based on how many protons came out so it's basically going to be an exchange of cations or anions depending what you have based on the affinity to retain on the column so we can say this is coupled with either cations or anions that will exchange for other cations or anions in the material passed through their mesh work all right and the fourth and last type that we talk about is size exclusion or gel chromatography so in this one your stationary phase is a gel having closely controlled pore size so you can control the pore size of the stationary phase and you can get different sizes and the different sizes basically will allow for extraction or separation of different sized particles whatever you're putting through it molecules are separated based on molecular size and shape when you're looking at these smaller molecules are temporarily retained in the pores this type of chromatography is really applicable to high molecular weight species so a lot of times when you're thinking of like things with really high molecular weight this is going to be what you would use all right any questions so far before we go on to a comparison of them or look at how they're used all right so looking at this diagram here we have the different types of chromatography we talked about so we have partition chromatography adsorption ion exchange and exclusion and so basically the type you want to use is going to be dependent on molecular weight as i mentioned that exclusion is commonly used with higher molecular weight whereas your lower molecular weights are going to stick to either typically absorption partition or ion exchange and then depending on what type of polarity you have do you have something that's non-polar polar and water soluble or do you have something that's ionic and water solid insoluble sorry and water soluble as to which type of chromatography you want to use so this is most widely used most widely use of all analytical separation techniques some advantages to liquid chromatography in more particular we're getting into high performance liquid chromatography is going to be the sensitivity you get increased sensitivity and we also have suitability for separating non-volatile species all right so we have a little bit more to talk about with just liquid chromatography in general and then we'll get into high performance of the chromatography instruments so i forget how many of you are doing liquid chromat or hplc versus gc for your papers next week you don't remember your papers you picked out i have no idea mine's the gas one do you see it okay i did so much work on with different articles for the uh paper for the class over brick that i can't remember what was what anymore okay and i know i looked at them too and i cannot remember i think there was some of both but i can't remember particulars all right so let's talk about some properties or different effects we see with liquid chromatography and the first one is looking at the effect of particle size of the packing so going back to the van diemer equation we have h equals a plus b over mu plus c mu that should be c mu all right so again what does the a represent in the van diemptor equation anybody what are the three types of band broadening do we have that we talked about one's eddie diffusion do you remember that no there's longitudinal that fusion or however you say that okay what's the third one what's that third type we have any diffusion longitudinal and mass transfer mass transfer all right so in order of the van diem dear equation a represents eddy diffusion which is independent of flow rate b represents your longitudinal diffusion which is inverse proportional flow rate mu represents flow rate and c represents your mass transfer which is directly proportional to flow rate we can rewrite this equation in regards to talking about packing and that rewrite is h equals a plus b over mu and the rewrite comes in from the c and now we write cs plus cm mu so in here cs is the concentration in the stationary phase and cm is the concentration in the mobile phase we can expand out cm where cm equals so cm mu is equal to f m times k prime i'm gonna go through each of these d squared p over d m and then all that's times mu so fm just means that it's a function of m the mobile phase k prime is the retention factor d the little d so i'm going to write it down here d represents the diameter of the packing big d represents diffusion coefficient coefficient fusion coefficient and then mu again represents your linear flow velocity or flow rate plus your flow rate and so here we have plate height versus linear velocity and so now we're looking at basically how do the two compare so your plate height as your plate height gets larger you have an increase in the linear flow velocity a reduction in particle size so if we have a reduction in particle size from let's say 45 to 6 micrometers so here's the 45 here's the 6 micrometers this results in a 10 fold decrease and plate height and remember going back to the relationship n equals l over h if h decreases then n increases and if n increases we have an increase in efficiency so again by reducing the particle size down that much it results in a 10-fold decrease in plate height which means ultimately you get an increase in efficiency so we can say that the efficiency of hplc column should improve dramatically as the particle size has decreased okay all right so the next factor for column efficiency and liquid chromatography is going to be extra column band broadening so in liquid chromatography you have significant band broadening sometimes occurring outside of the column packing itself this leads to extra column band broadening and so you get extra band broadening as a result of the location of the packing bronyin arises from differences in flow rates between layers of liquid adjacent to the wall and center of the tube so we can look at the relationship and equation for this to figure out our efficiency so we have h e x is equal to pi r squared mu divided by 24 d m where h e x represents the contribution of extra column effects to plate height dm again is that diffusion diffusion coefficient of the solute in the mobile phase and that has units of centimeters squared per second next we have r where r represents the radius of the tube and that's given in centimeters and then mu again is your linear flow rate or linear flow velocity and that's given in centimeters per second so here we're looking at what effect ultimately the radius would have on efficiency so let's look at the radius right here which is directly proportional to h so if the radius decreases what happens to h will it decrease too yup it would decrease so if r decreases h would decrease let me erase the line i don't know why that does that okay and if h decreases what does that mean for n remember we have n equals l over h so if h decreases what happens to n it increases it increases so r and h in this first equation are directly proportional h and n are inversely proportional so by decreasing the plate height we allow for an increase in efficiency because if n goes up then efficiency increases so we can say a reduction of the radius improves efficiency all right any questions before i go on to the third one all right okay i see diane now all right let's talk about the effect of sample size on column efficiency and so again we're relating that efficiency to the plate height so if we know the plate height change looks like we can tell what efficiency is going to happen so here we have the different partition k prime values and what we see is that as size increases we see an increase in h so these partitions are representing ultimately the size effective relative yeah that's representing the size so as the size increases plate height also increases if plate height increases what does that mean for n does it decrease it decreases and if n decreases what does that mean for efficiency it becomes less efficient yes efficiency decreases as well good so we can say that efficiency always decreases as the sample size is increased all right so we can look at the effect of increasing the sample size so effect of increasing sample size on efficiency efficiency well that's that's a why something like that um is increased as tr the retention time is increased and this is because late eluding peaks have a lower efficiency so we know those late eluding peaks have lower efficiency which means if those have a lower efficiency they're going to have a higher plate height and it's the result of an increase in the sample size all right so the next and last part of the lecture is going to be focusing on the instrument components of hplc so whatever we don't get through today um will be what i'll be recording for monday um so we'll have the rest of this lecture for monday then you'll have your presentations on wednesday finish up on friday the following monday will be your exam so we have a little bit of lecture left before we go virtual then and we'll do the rest when we go virtual all right so this is the general overview of what an hplc looks like so here is your inductor valve so you're actually going to have two different types of columns and typically we have these two columns one's called an analytical column and one's called guard column the guard column is put in place to protect your analytical column these columns are really expensive and if you have something going to your column that is not what you want then it basically will destroy the analytical column the guard columns are much cheaper and so ultimately we look at if we have to destroy a column have it be the guard so we will talk more about those columns but your sample is injected and you have a pressure transducer and you have a back pressure regulator so ultimately you have a bunch of stuff going on here so here's different solvents you have a pump you have your check valves whatever it might be so we'll talk about each of these components the hplc instrument is a lot more um in-depth than a gc or any other instruments we've talked about but we will look at each of those all right so let's start by the solvent reservoir let's start by looking at the solvents that are in an hplc so here's your solvent reservoir this is going to be mobile phase reservoirs and solvent treatment systems so you have the solvents here you have getting things thrown at my door sorry um you have different types of solvent treatments and this is going to be basically a waste container so you have your sparger so right here is the sparger and the sparger is in place to remove any dissolved gases so any dissolved gases that come in play will be removed through the sparger you don't want excess gas going through to the detector because it will destroy it and so this is going to basically you sparge your sample or your solvents actually to make sure you don't have that air that gas in there and then right next to it we have the inlet filter and so that is going to be a filter to dust to filter out dust and particulate matter from the solvents the solvents that are used for hplc are reagent grade so they're they're a little bit more expensive but you want to make sure that any type of particulates from that are filtered out along with any of the gases before you proceed this process only takes place once once you put a new solvent in because it's a sealed system but after that each time it will clean those solvents all right so with this we're going to get we're looking at two types of elution we see we see isocratic and gradient elusion so for isochronic illusion we have separation that employs a single solvent of constant composition whereas gradient dilution is going to be two or more solvent systems that differ significantly in polarity so you can use either a single solvent system or two or three solvent system by doing the two or three solvent system you will see an improvement so it improves separation efficiency and the way it does this is after illusion begins so after your peaks start showing up the ratio of solvents are varied in a programmed way so none of this is manual it's all done internally on the instrument um and it will control that so that it's a uniform change each time as you will see as we talk about this there's a lot of conditions you can change and a lot of things you can do that are going to give you different type of values so it's really important when you talk about hplc or you observe hplc that these conditions are identified you need to know what type of evolution you have you need to know type of columns you're using you need to know what type of um conditions were matched so these are things to think about when you guys are reading your paper to look through that yes mindy sorry i forgot i was muted um what does that last line say at the bottom after illusion begins ratio of solvents are varied in a programmed way yeah i realize the writing kind of blurs together sometimes on here i also apparently have bad handwriting and sometimes and my kids learning how to read and i don't clarify my letters and it confuses her so all right so let's look at the advantage to gradient illusion because i'm going to say well i mean you would think why won't you just use it well obviously there's a cost associated with that so there is some advantages to it um so here's an example of this one's showing isocratic illusion and this one's showing you your gradient illusion so for advantage of gradient dilution is that gradient dilution is shortened separation time significantly without sacrifice and resolution of early peaks so you'll notice that we still see the same 10 peaks but we went from about 30 minutes total down to less than 15 and you still see all 10 peaks this is similar so similar effects to what you would see in gc gas chromatography with temperature programming all right questions on that