Ion exchange chromatography | cation exchange chromatography and anion exchange chromatography

welcome back friends welcome to another video tutorial from Thomaz Bellucci and in this video tutorial we'll be talking about ion exchange chromatography we've been talking about chromatography for many videos and in this video we'll be talking about the polar chromatography or the chromatography for the molecules which are polar in nature you know chromatography is the separation of molecules from each other now the molecules could be of different types there could be macro molecules like proteins nucleic acids or there could be smaller molecules like ions now in this case of ion exchange chromatography we separate charged molecules and polar molecules okay now hydrophobic molecules cannot be separated by this method now normally when you look at chromatography I always told you in my earlier video regarding the chromatography basics and also the types of chromatography there in chromatography you need to keep certain things in your mind in all these cases of chromatography there are the interaction between a stationary phase and a liquid phase and a mobile phase okay so two different phases are out there one is known as a stationary phase and another phase is known as the mobile phase stationary phase is stagnant it is fixed while the mobile phase is moving in either direction whatever you run the chromatography but it is moving this is the mobile phase these are the two different phases that always interact with each other now the interaction between stationary phase and mobile phase varies for different molecules and this varying nature of different molecules interaction with stationary mobile phase will ultimately provide us the information about knowing which molecule will separate fast and which molecule will separate slower now this is the interaction between these two phases that determines and that ultimately causes certain molecules to elute and come out fast certain molecules to eluting later that is the actual concept of chromatographic techniques it is the phase separation methodology that we use now in case of ion exchange chromatography what kind of molecule will separate ions or polar or charged molecules so we always go for charged or polar molecules in this case now in this case of ion exchange chromatography we run a column you know we also talked about column chromatography where column chromatography means we prepare columns in it's a structure made with polymers either could be of glass or it could be of any of fiber polymers that creates a chamber inside the chamber we add the stationary phase okay the stationary phase can be of different molecules those are polymer molecules like cellulose and like you can use here agarose so those are the molecules which are polymers and the polymers actually can create networks and mesh like structures where all the other molecules can move through those networks so that's why we need this stationary phase as polymers now in this case of ion exchange chromatography the stationary phase that we use is it is in this case either cellulose or agarose use both either cellulose or agarose you know both are very common I hope you know all these things now in this case of ion exchange chromatography the idea is the exchanging this charged or polar molecules from the mobile phase because here the stationary phase is solid which is say other rows for example this is solid while the mobile phase here is liquid remember this two phase can be of different type like it could be liquid or gas okay but here the stationary phase is solid the mobile phase is liquid now the liquid contains all other molecules among them we have the charged molecules and polar molecules that we want to separate from each other and in this phase we have agarose so what we do here we take this agarose and let us assume this as X and we attach ion with it some sort of ion or charged molecule with you now the idea of ion exchange is to exchange a specific polar molecules okay now there are two different types of ion exchange chromatography one is the cation exchange chromatography another one is the anion exchange chromatography in the cation exchange chromatography they retain cation in the anion exchange chromatography this column retain an ion that is the idea that means the stationary phase is fixed this is the stationary phase this is the fixed stationary phase and if it is a cation exchange chromatography in that case this will attach to a cation okay so let's say cation means positively charged ions so let us say here C is the molecule that we want to separate from the mixture it is in the mobile phase remember this is in the mobile phase okay and this is in the stationary phase now C is positively charged it's a cation that we want to separate and here we have the agarose attached with a molecule it will be attached with say with say this so this is the construction what is mean this is the agarose attached with an ion okay and that ion is blocked by another iron of same type okay maybe it's a cation exchange chromatography what it means this will exchange this cation for the other one so let's assume here in this mixture we have both cations as well as anions let's draw another anion here then ion say D D - so we have a cation we have an anion this is the mixture from this mixture we only want to separate cations okay and this is our stationary phase where we have dagger rows attached with the ion exchanging molecules the negatively charged molecule which is fixed with this with this agarose as well as we have a positively charged molecule which is masking the negatively charged molecule there now the idea here is the exchange of iron how the exchange will work remember this part is completely fixed as stationary so the exchange will occur between this and here right so here how the exchange will happen as this Annerose is attached to a negatively charged the molecule negatively charged molecule will always try to interact with a positively charged ion so among this mixture of negative and positive this a will try to pair with c plus instead of D because D and a is the same charge they will repulse where C and a opposite charge will attract so after that what we can get is something like this now it will be attached with the C plus so now in the stationary phase we have C plus attached while the B plus and D minus will be now present in the mobile phase and this thing will be eluted this will be eluted after that okay so this is the idea the big picture this is how it works ion exchange that means there is ion present and there is one I am attached and you will exchange that attached ion for another ion that is in a mobile phase that is the idea now this is the cation exchange remember it is a cation exchange which is positively charged now if I look at for the anion exchange or negatively charged the case will be like this in the in the anion exchange chromatography the raising the raising means the solid phase that is present in the column the raising we contain positive charged molecules instead of negative because here they need to bind with anions okay and then of act with they also present a negatively charged attached to it then in the mobile phase let's say same things are present in the mobile phase d- + c + so now what we know as disease the stationary phase the fixed phase and it contains a positive charged ion it will interact with this negatively charged ion in the mobile phase so now the pairing will look something like this a + along with that we will have this D - why the C + and this B - this is the ion it will come with the mobile phase so remember this D - my letters to the a similarly here as a is negative C + attached to the a so ultimately after that it will be eluted so this is the idea okay it will be removed not actually illusion its known an illusion or removal after this removal is done remember after running this whole system after running this whole process whole column with all the solutions and molecules what we will get we will get some things like this where we have the agarose bind with our molecule of interest okay now all the unbound molecules are washed away with buffer solution once that thing is done then the idea is to separate these molecules independently because we want them we want to fish them out from the mixture of other ion molecules so then after that what will we do we exclude these molecules that process is also known as Ellucian ok and how we do that we simply increase the concentration of same type of or same charged ions in the buffer in the solution we increase the same charged ions ok for example here we need the positive charged ion to elute this one so we increase a particular positive ion concentration hugely so that those ion start to bind with this and see start to come out this is the way we elude things so for any chromatographic reaction that you want to see in future keep three things in your mind first thing you need to know about its stationary phase then you need to know about its mobile Fay's and how the interact second thing is that what kind of solvent they are using for this process like in this case we use activist molecules with hydrophilic molecules or charged molecules or polar molecules in this case organic solvents are generally not being used though we can use this process for many different varieties that I am going to talk you later but this is the idea this is the first thing you need to know the second thing you need to know is the principle of how it's working how the separation working in big scale and the third thing you need to know is how the illusion take place that means how you separate your target molecules from the agarose binding or from from the stationary phase after the chromatography is complete so once you know three things you know the complete overview of that chromatography now as I told you all these three things now it's time to talk about the advantages and disadvantages of ion exchange chromatography the advantage is that an exchange chromatography works for polar molecules and charged molecules so very well and we use ion exchange chromatography in the field of water testing plants where we need to check for certain water or water dissolved molecules and separate them from each other so it's used in water testing labs in you know in different wastewater treatment plants and stuff second thing we can use ion exchange chromatography for separation of proteins for example we can use it for the separation of hemoglobins as well as different power firings we can also use it for the other poor firing ring containing molecules for example like chlorophyll also so these are the advantages of an exchange chromatography however the disadvantage is that in this type of technique we cannot use the those hydrophobic molecules for the separation it only relies on the charge and as it is relying on the charge a single charge change can cause this whole process to change an alter you know and amino acid you know protein is consisting of many different types of amino acids right so all these amino acids have different charges right now if one of the amino charge changes that will ultimately change the overall charge of the protein and that will affect in the separation of that protein using ion exchange chromatography and this is also known as electro folk Okemo chromatic focusing it's known as chroma - focusing chromatic focusing means changing in one small charge in a molecule in in proteins mainly will lead to the change of the overall charge of that molecule and by changing this one charge we can modulate the illusion of that molecule using ion exchange chromatography so because you know this illusion is very very important how you ultimately get the illu tent and the molecules at the end because you need to have all these molecules at your hand so that's your idea you need to purify it and then you need to use it for your purpose so here that is called as a chromatic focusing step okay for focusing a specific charge or focus on a process where we get a specific charge molecules there and by changing that charge you can get the details by changing the charge you can get that molecule out from the column pretty easily that is the idea about the ion exchange chromatography I hope you understand an exchange chromatography if you liked the video please hit the like button share this video with your friends and definitely subscribe to my channel to get more videos like that thank you

Channel: Shomu's Biology

Views: 337,770

Keywords: suman bhattacharjee, shomus biology, biology courses, life science, Ion exchange chromatography, chromatography, cation exchange chromatography, anion exchange chromatography, ion exchange resin, protein chromatography, column chromatography, ion chromatography, chromatography lecture, ion exchange chromatography lecture

Id: p3_WtEYIhTo

Length: 14min 59sec (899 seconds)

Published: Mon Jan 18 2016