Thin layer chromatography (TLC) principle explained

friends welcome to another video tutorial from throws gravity and in this video tutorial I'm going to talk about thin layer chromatography also known as TLC okay slip stop now we talked about many different types of chromatographic techniques but TLC's was the very basic type of chromatography and actually little bit advanced version of people chromatography and even in the baseline of other chromatography which are quantitative like gas chromatograph or a liquid chromatography thin layer chromatography works in a very similar fashion with that of the people chromatography now to understand thin layer chromatography you need to understand the basic mechanism of the people chromatography so what if people chromatography chromatography are techniques which help us to separate mixture of different substances based on their mobility because in any type of chromatography we have two things in common it contains a stationary phase it also contains a devices now stationary phase which is fixed and mobile phase is moving so mobile phase carrying our substance will start migrating all the mixture of substances throughout the stationary phase or diffusing the stationary phase right so due to the differential migration rate of all those substances or molecules few of them migrate further few of the migrate less that's how we separate different substances from mixture that's the idea of a paper chromatography okay so the same idea applies here you can help yes your thin layer chromatography so let me write down some things we have stationary phase and we have mobile phase Swink is of this TLC or thin layer chromatography we also choose specially present mobile phase depending upon the type of substance that we want to separate from it okay now the major difference in terms of TLC with liquid chromatography if TLC's or so paper chromatography but this is a little bit modified like because in paper chromatography the matrix on which the chromatographic take this is only of paper that's it but in case of PLC it carry the stationary we reaches stationary phase which is mostly made up with either plastic or silica or any other inert material or using hand that part we do whether it's a plastic or silica is like a food on top of aluminium foil that's the difference between the create depth we use for VLC and the day that we use for people chromatography okay so it's an aluminium foil on top of which silica is applied as a put that's what that means acting as a stationary phase that silica is acting as a stationary phase for most of the senior chromatographic speakers on the other hand of mobile phase is most of the time norm wouldn't because you know silica is a cool element so generally we chose non polar mobile phase if we have a stationary phase which is polar and if you have a non polar stationary field then use the polar moment okay but most of the time position if I use here is another one and then the mobile phase you check is unknown input that's what we use in case of this serious chromatography now why it is called thin layer because normally the silica is coated over the aluminium foil but this coat is very very thick short thick the reason behind this idea of making a very thin layer is that this thin layer will help to resolve all the mixture of molecules from each other farther and faster compared to the thicker because if a layer is thick in that case the mobile phase will be trapped and absorbed by the stationary phase more often but if the air is very thin then the mobile phase will not interact with the stationary phase if we do not get absorbed by is much that is idea behind making the place P f we have Celia schematically now the question is why we use a layer chromatography now in any other chromatographic technique that we use we generally use them to separate different molecules from it same thing applies here in thin layer chromatography mostly we do thin layer chromatography to taste the presence of any toxic substance you know food for like any pesticides or insecticides in the food that we can easily find out all they say we extract a plank of material so plant extract and from the plant extract we want to separate the different components of the plant extract let's say from very from a medicinal plant we want to separate all the components of the protection that we can use use easily with helium now the question is how thin layer chromatography is performed and what is the exact mechanism behind the thin layer chromatography so people talking about the mechanism I must tell you how its performed what's being happier than failure from an already simply we have a glass vessel a big glass poppy and we can't because the silver they put we put the mobile field this is the moment a very big moment this is a disease non-polar mobile face the good is okay and then we have that TLC plate so what we do generally we take the TLC plate and if it is like a slice of the place up it is normally big you can take a slice of it generally you simply take the height depending upon the the substance that you use the mixture that we use to separate generally the plate can be let's say five ten centimeters long you can take so if it takes its place and you simply take a modest pencil in this case approximately 1.5 50 difference now this is the part it's like a running start point it's like everywhere we start running we all start for it an end point similarly this is the start point and let's say you you must forget come out to be an infant it's the one centimeter effort about the end of the clip so the start point is somewhere you put 50 point when we apply the mixture of solution that you want to separate from it okay so this is a sample application spot which we applied by putting some samples in and you want the championship to learn from the start point to be that's what your IDs now how exactly your sensor will run from start point to the end point by putting display into the by this because stationary phase is fixed it will not separate the sample that you put there the only thing that can separate your sample is the mobile phase right because all the such organ that you use in the mobile phase will drag your sample or specimen to the 8.00 thing that's right now you put this place here so let's say this is this is a point the start point and yes should we apply the this meat here and we put it bit it actually will be sweet we put it a little above the solute that's already there because we go we should not submerge it in the solvent directly we allow the Fondren to to be Ricky less in length from the start point and actually when we apply the sample first we made this TLC plate completely dry with sheet so that no further division take place so the circle is properly fixed into the station is now we put the plate into the mobile phase and then we close the lead of the glass so now it's time to allow the mobile phase taking all the substances for the finish line how they do that with the help of capillary action we will be grave of the capillary action the solvent here try to move all the substances the mixture of substances from the star point to the end point actually the end point is recently less than the end of that will flip because you will not allow them to completely run out of the plane right so you to make sure that if you stop the process once it reaches a specific deadline next we apply the finish line so then we allow them to diffuse and that's what happens slowly all this water will start by heading towards the finish line no more white in this migrate across the piece now the question is what is the exact mechanism of taking the mixture towards the finish line we keep depending on the differential rate of absent through the stationary phase by the solvent that means the mixture of specimen that is present here as a solvent those two things complete for each other or the migration through the stationary piece that's what happens now outright emulsion hunting let's say this molecule stirs the specimen that you provide here the sample if the sample nature is very similar with that of the stationary phase then what will be the mobile phase we move faster because this sample is if the the quality of your sample is interacting with the station it is more it will move slowly as if the sample is not interacting with the stationary phase that it will move faster with the help of the Sun that is the episode you do that few of those business we move faster and further few we would listen and that's how we end up the different spots in the CFC plate so this spot like here Defense Force energy like sport means you know once the process is run you may not see this one like it can if you may not be visible at the very beginning so you need to make sure and you can visualize the spot later so three things matter here during the separation what is the nature of the solvent the nature of the stationary phase and the sample that is used the nature of the solvent and nature of the stationary field should never be the same because we those things are the same then it will not allow all the separations on that's why if we use the polar stationary phase we should not use a polar go by this you should always use a non polar that's what we do but you never know about the sample that it it can be polar or noble so depending upon what kind of material it is depending upon which it will be separated that so now what we do once the process is done it reaches its peak that night what we can major axis we can we can actually see the place to be likely absorb with the solvent or not a little visualize that some part we'd already reach this the finish line because there is no color so this is a very important step to to look at it every single time all the experiment is going on that we need to stop the process while it reaches the edge line we will take it up is right so upset the drag is done then what you do you want to develop the field is also like a pill because you need to make sure where exactly we get the spots because that's going to tell you the separations of the specimen now how can you develop spots because we cannot visualize them on it like a color so answer to that is present even before you started the experiment so what you keep normally the silica that is coated over there a media file if if it generally contains these up it contains zinc sulfide zinc sulfide is like a phosphor and the nature of phosphorus whenever you apply UV light then what do you see you see dark spots why because the complete TLC plate contains the first book which will give you a color in presence of UV light but whenever there is a presence of it the chemical factor those regions of the TLC plate we say to give you the color the light so whenever you put that PSD plate in Yui after the process is done you will see dark spots then if those are the regions where the phosphor is not present because the the diffidence is only occupying those regions that's why there is no fuss for presently - mine is the task spot so whenever you are looking it under you need you will see a dark spots and those dark spots are the regions where your specimen is actually present so by asking at this TLC plate you can easily tell that yes this and this are the two separate mixing that is present in your mixture that's how we can separate them now the question is this process is going to tell you about what are the constituent of a mixture now the question is so it is qualitative or quantitative non sorry it is quantitative you can quantify the presence and the concentration of each of the specimen how for a quantitative and say we need to calculate something right so what is the calculation here the calculation here is made by a simple idea that the total distance traveled by each of the four divided by so distance troubled by each spot divided by the total distance covered by the solvent and this is known as RF value a retention factor so our Y value is going to give you an idea about the migration they have separation differential migration rate all those substances that are present in the mixture increases on specific mu by six because if you change the mobile phase are in very meeting if you change the stationary phase are in can you reach it but for specific stationary Taylor a mobile phase now I bet about different sampling specimens will be different that will help you to understand what kind of spins when you're dealing with so with this idea you can also separate even smaller tiny differences between different molecules that's the beauty XY easy to calculate because it simply take this and once you see the spot you mask them you mark them with pencil right because you know the colors may trade you may not see the feature so you mark them can you take the spread out and then you measure the length with simple Google scale simply measure it in centimeters and the total is already linked you also majors in centimeter you divide them to get the RF value now how is that is quantified to quantify the presence of specimens you need to learn this same experiment with low concentration of a sample of the pencil so Phi the standard curve so once you get a standard curve with known concentration of specimen then you run the mountain additional specimen you can easily take or you can easily identify and quantify the amount of a specimen that is present that that's it that's what you can do okay another way that we can find out the presence of the separated components of the specimen is by different chemical processes because you know this is the process where you use a function in some other processes are there we don't use phosphor but any chemical things like cells giving athlete can be taken as a phosphor in most of the cases we're getting the presence of a specimen that wictred darkest because otherwise the silica the important task but wherever you will physically present those regions will turn darker so you get a dashpot present when we expose the TLC plate in presence of the traffic so that's how you get different spots out and from the spot again you can measure protein now the question is those thoughts are like circles so when you are measuring the distance how can you exactly measure so this is circle which part you should pick the top bottom of the fender now the answer to that if you get a funnel you also want to get a slicker of that circle and whenever you needle in the distance you will always measure the distance from the start light to the center of the circle that's all you should please so that's how we get the place of PLC from the DL state we can quantify the presence of difference visibility concentrations so that's how we grow more TLC it's very basic technical very important technique on the separation of many real-life examples that I told you like a separation Mahajan finding toxic substances present in your food and water supply things like that so if you liked this video please hit the like button share this video with your friends and subscribe to my channel to get more and more different

Channel: Shomu's Biology

Views: 320,372

Keywords: suman bhattacharjee, shomus biology, thin layer chromatography, thin layer chromatography explained, thin layer chromatography tutorial, thin layer chromatography rf, tlc, chromatography, thin-layer chromatography, chemistry, organic, thin layer, chromatography explained, chromatography paper

Id: CtOFzh4oVCU

Length: 18min 44sec (1124 seconds)

Published: Wed Jul 05 2017