Column chromatography - gel filtration chromatography lecture

friends welcome back to another video from somos fallacy and in this video tutorial we'll be talking about size exclusion chromatography we've been talking about different types of chromatography lately and in this video I'll be talking about the size exclusion chromatography size-exclusion chromatography is also known as molecular exclusion chromatography or molecular permeation chromatography it is also known as molecular exclusion chromatography so all these names molecule exclusion chromatography gel filtration chromatography size-exclusion chromatography molecular exclusion chromatography all of them are the same this is the first thing you should know all of them are the name of the same technique that we know so here I will talk it about this process now it is also known as gel filtration I told you gel filtration chromatography now if you heard this name gel filtration chromatography it's much more clear to understand about the technique so why it's called as gel filtration gel filtration means here filtration means the filtering of molecules because you know chromatography means separation of molecules from each other it may be depending upon their charge it may be depending upon their size and mass and all the stuff now we already talked about the ion exchange chromatography where the molecules are separated based on that charge in this case of gel filtration chromatography or it's also known as molecular exclusion chromatography in this case we separate proteins based on their size and molecular weight the molecular telling molecular weight is not perfectly okay here usually they separate molecules based on their size okay and so this is the parameter based on the size of the molecule so as its size based molecule we mostly use this technique for protein separation as well as nucleotide separation mainly for the protein separation like any other any other chromatographic techniques we need to know the basic things that is what is the stationary phase of this what is the mobile phase of this and what is the column means and how its prepared the idea here the stationary phase for this size expression chromatography is agarose you know polymer network that can produce network between each other you know agarose can have small pores prepared between them like this small pores are created now if I draw the column the column is the chamber where we put all the stationary phase molecules there this is the column and this column is filled with filled with all those mash networks throughout let us say throughout this network is present okay now here this networks contains pores in it so we need to prepare this column or stationary phase with some polymer molecules that can create this net-like structure now the molecule here we use is agarose so agarose can create this type of pores we know that because we can separate DNA using agarose using gel electrophoresis and stuff so the situation of this the process of size exclusion chromatography is very familiar with the process of electrophoresis but there is a significant amount of difference while in electrophoresis we drive energy we create an energy barrier with which a voltage gradient with which when the molecule is moving the force is created and provided by the change in charge that we apply the current flow that we apply but in this case of the size exclusion chromatography we do not need to apply any voltage it is entirely based on the gravitation force that the molecules will come that is a significant amount of difference between the size exclusion chromatography and the gel electrophoresis remember that so here it is by the force the driving force by the gravity okay so now let us say this is the column and we prepared the stationery phase with agarose now what we do we load this with the molecules the mixture from where we want to separate our molecules now the mixture contains various size of molecules let us say the mixture is homogeneous it's made with proteins only but some proteins are smaller some proteins are larger so here the idea the principle of separation is that as they are creating the pores the pores are very small so the large protein molecules cannot enter inside the core okay they cannot enter inside the pol as they are unable to insert inside the pore they can move through other regions other regions through this because remember in this case production of this column is not rigid like that this column has many blank spaces in it and this whole volume of the column is a column volume so column volume is nothing but the solvent volume okay whatever solvent we apply that is the volume of the column that is the idea but there is another volume some regions are blank through which the large molecules can pass easily because the large molecules cannot enter inside each of the pores because pore sizes are smaller only small molecules can insert inside each of the pores so as the small molecule will insert inside the pore and it will move through the pore it will take more time for the small molecules to come out while the large molecules will come out pretty fast because they will be traveling less distance because they are which are very less volume here so the volume which is covered by the large covered by the large molecules is known as void volume void volume that is the volume which is the volume where the smaller molecules never enter the volume consisting of the large molecules moving through the column that is the void volume while the column volume is so volume the total solvent volume so this is the idea if I draw you another picture it will be much more clear let us say here this is one specific pour if i zoom into one of the pores it will look something like this and in this pour let us say there is a small molecule and there is a large molecule so what will happen this large molecule cannot enter into the pore so it can easily pass through this but the small molecule will enter and it will pass through this whole chamber so it will be entrapped smaller molecules will be entrapped inside those pores inside those chambers so as they're in trapped inside it will take long time for them to come out and the large molecules are not entrapped so they will come out pretty easily earlier okay that is how you separate things you might think that small molecules will travel fast large will travel later but no here it is not the case yet the case is small will be trapped so it will come later large will will not trap so it will come earlier in the illusion stage now the illusion means after you load this column with those mixtures then you run buffers and after some time you start taking out molecules this because the solutions will slowly come down using the force gravitation gravitation it will come down to the bottom and then slowly you start collecting all these molecules in chambers okay collecting living chambers now you start collecting larger molecules first so if I draw the chamber of collection in this way let us say we have 1 2 3 3 different chambers let us say at the very first chamber we get the larger molecules coming out the second chamber we get the moderate-sized in the third chamber we get smaller size so that is the idea if you look at the time duration in the time gap you will get a curve like this you'll get a curve let me draw it this this is time the x-axis this is the rate of Illusion the y-axis so how it will look like it will look like this this red is the big one so big one will elute first while the smaller one will in later so this is for the small this is for the big this is how the illusion take place okay this is the graph it will look like so this is the idea of size exclusion chromatography or gel filtration chromatography or whatever you say the molecular exclusion chromatography is the entrapment technique that we use very similar with the technique called gel electrophoresis but the difference is the driving force here is gravity where the driving force in in case of the gel electrophoresis is the current flow so that I hope this is helpful so let me talk about a little about the advantage and disadvantage before closing the advantage is that we vigorously use this technique for separation of larger molecules all the time is very common technique to use and the separate things based on their you know based on their size and another thing which I should tell you here is about the hydrodynamic volume they actually separate molecules based on their hydrodynamic volume hydrodynamic volume is the area or the volume taken by a molecule which is present in water or solution that is known as the hydrodynamic volume so as they can separate molecule based on their hydrodynamic volume it is this technique is very very important to think of separation of an identification of both folded and unfolded protein it can get an idea of whether the protein you are dealing with is folded or unfolded because the hydrodynamic volume let me write the hydrodynamic volume completely filled so let's erase some of the stuff hydrodynamic volume the hydrodynamic volume that we are talking about hydrodynamic volume for a folded protein and unfolded protein for a folded protein it is 14 angstrom for an unfolded protein it is 36 angstrom so you know degree so this is the idea the hydrodynamic volume for a folded protein 14 for the unfolded 36 because you know the unfolded protein will occupy more volume because it's scattered it is unfolded but a folded we are Q less area so this is the idea so we can easily separate folded and unfolded protein using this technique so we can tell whether the protein we are dealing with is in its native state or its unfolded state that is another very important advantage now some disadvantages as this process applies only separation based on the size and hydrodynamic volume it is not very good and the resolution of this process is not very good though the resolution depends on resolution means how we separate molecules and how closely the size of the molecules could be to separate them that will be known as resolution in this case now the resolution for this is very it's not good because here for this column to run it depends on the type the percentage of agarose that you take the more percentage of agarose that will take the more smaller the pore size will be and so the separation will be different so these things matter for the separation so this process is not very high resolving process with low resolution that's one disadvantage another disadvantage is they cannot separate proteins based or their molecular weight it's not always true you read that it can separate by molecular weight but it is not actually based on the molecular weight it is not completely true and the third thing is that for same type of molecules it's very difficult if you have same very very close size difference it's very difficult for for this technique to separate them from each other so these are the advantages and disadvantages of size exclusion chromatography or molecule exclusion chromatography or gel filtration chromatography whatever name you want to say and I hope this video helped you if you like this video please hit the like button hit the subscribe button that is present here in the top as well as in the bottom and subscribe to my channel to get more videos like that thank you

Channel: Shomu's Biology

Views: 132,593

Keywords: suman bhattacharjee, shomus biology, molecular exclusion chromatography, gel filtration chromatography, gel permeation chromatography, size exclusion chromatography, chromatography, size-exclusion chromatography, column chromatography, protein purification, liquid chromatography, size exclusion, size exclusion chromatography protein, size exclusion chromatography principle, Column chromatography, gel filtration chromatography lecture

Id: eH7UkTB7m8U

Length: 14min 6sec (846 seconds)

Published: Mon Jan 18 2016