you [Music] [Music] [Music] [Music] hello students hello students today I am going to show you how to purify a mixture of compounds using column chromatography column chromatography is a powerful separation technique which has wide applications in various branches of chemistry biology and pharmaceutics which have wide applications again in industry before I start this technique let me ask you whether you know any separation techniques yes distillation sublimation regress realization good if there is a mixture that has two or more components that have similar properties which cannot be purified by the separation techniques that you mentioned we use column chromatography so column chromatography in which we actually observe the compounds onto solid stationary phase and purify it using liquid mobile phase so a chanela phase and mobile phase here good question stationary phase is usually silica gel onto which the compounds are actually adsorbed and mobile phase is actually a solvent or solvent mixture which we use to separate the compounds however we learn more while we are performing the experiment sir you have used the word adsorption twice yes can you please explain it adds or PI nomina in which a compound is physically or chemically attached to a material in this case the compounds are actually attached to or adsorbed to silica gel so column chromatography is a part of adsorption chromatography so adsorption chromatography is classified into two things one is called thin layer chromatography we call it TLC and column chromatography okay students today I will show you how to perform column chromatography to separate the mixture of compounds containing and yellow compound and which is component a in this case and a red compound which is compound B in this case okay all right so column chromatography basically involves a glass tube packed with a slurry of silica gel which is a stationary phase and the loaded mixture will be separated into components based on adsorption and polarity but first of all you need to perform thin layer chromatography which we call it TLC to obtain a suitable mobile phase to separate the components of mixture pretty well students this is a TLC I took a mixture of compounds a yellow one and the red one however I am not going to show you the details of thin layer chromatography today but this experiment suggested me that I can use a t20 mixture of hexane and ethyl acetate to purify the compound in addition it also suggested that I can load the column with hundred percent exists and increase the polarity to separate the compounds this process is called illusion so if we can purify the compounds using TLC itself why do we need column chromatography good question TLC is usually used for milligrams of compound whereas column chromatography is used for grams of compounds okay so students as I showed you today this is TLC is a sheet of silica gel I'm going to use the same silica gel for column chromatography which follows the same principles of TLC so students you can see that I have fixed the column to the stand and tighten the screw so that the column doesn't fall down and break I left some space at the bottom so that I can collect the compounds by putting the beaker or a test tube at the bottom of the column okay alright now I'm going to lower the column but you know that you can see that silica gel is a fine powder and it will pass through the column so I have to put cotton at the bottom of the column press it tightly so that the silica gel doesn't filter through it and I'm going to do it right now [Music] so students I have used the glass red to press the carton tightly now I'm taking the glass rod and keeping it outside now I will tell you how to make the slurry of silica gel but before doing that I need to wear the safety goggles and safety gloves so that the silica gel that I am going to take doesn't fall into my eyes or onto my hands okay all right so I am going to take a beaker and some of the silica gel that I already showed you then I am going to take some hexans which I have it here and pour in it now I take a glass rod and stir it nicely so that I can make a slurry of silica gel okay sir why are you staring it Oh we stir it in order to get rid of the air bubbles and also the silica gel will mix uniformly with the solvent okay okay so students have made the slurry of silica gel and how to load this slurry of silica gel into the column but before doing that I need to put a little bit of solvent into the column how to put it very carefully so that some of the solvent is right above the cotton okay now I'm going to load this slurry of silica gel using a glass funnel okay I am putting the glass funnel on the top of the column nicely so now at this part you have to do it couple of times okay then we can collect the solvent then you can pour it back then you can open the stopcock like this and then you can collect the solvent make sure that most of the silica gel is already loaded into the column now I'm putting this beaker aside now I'm taking a glass pipette and I'm going to wash the sides of the funnel and the column using the exists that I'm going to use but before that I'll put a beaker at the bottom so that the solvent doesn't fall on the floor okay so I'm washing the sides of the funnel so nicely now I'm taking out the glass funnel because totally the silica gel and the glass funnel is washed out now I'll take some of the hexanes and wash you can see it carefully that I am washing the sides of the column so nicely that the silica gel that is stuck to the column sites has been washed washed inside so now I'm going to release the stopcock very gently so that the extra solvent which is on the top is going to come down slowly then the silica gel is settled down nicely there is some solvent on the top of the silica gel make about three to four inches height of silica gel so that the compounds can be separated properly so how did you determine the height of the column there are many factors to determine the height of the column but most importantly it depends on the amount of the compound that you want to purify okay now students you can see that the silica gel is settled nicely and there is some heads ends on the top of the silica gel now I am going to tap kaanum sir why do we need to tap the : o we need to tap the column so that we can get rid of the air bubbles and the silica gel will settle nicely and the purification can happen properly okay sir now you tap the column a few times and then release the stopcock and let the solvent pause by then you close it again you tap it nicely so that it settle down Eisley and then again you open it then one last time you can tap it nicely and make sure that the column or the silica gel is packed very nicely you can repeat this three to four times so that the silica gel can be packed properly okay now close the stopcock and let's do that experiment [Music] [Music] [Music] [Applause] [Music] [Applause] [Music] [Applause] [Music] so students now I'm going to take the mixture of compounds which I am going to show it to you here and add some chloroform to dissolve them and some silica gel to make a fine powder so first of all I am going to take some chloroform very very little bit and I am going to add it to them so that they are dissolved make sure that they are almost and completely dissolved then add some silica gel to it so that the mixture is completely adsorbed onto the silica gel so in order to evaporate the chloroform I am going to take a glass rod and I am going to stir it nicely so that the compound will be completely a fine powder students this will take at least 5 to 10 minutes in order to get a fine powder [Music] now students you can see that after steering it for five to ten minutes I got a very fine powder now I'm going to load this compound onto the column but before doing that I am going to put some hexanes into the column using a pipette please be careful while you are pouring the exams into the column pour it uniformly so that it doesn't disturb the top layer I'll pour little bit more now you can tap the column if you want to make it a nice layer now I'm going to load the compound using the funnel so I'm going to put the funnel on the top of the column and I'm going to slowly pour the compound into the column so that it can settle down nicely so you can see that I almost finished all the mixture yes now I'm going to take little bit of more hexanes and I'm going to wash the funnel if some compound is stuck to the funnel then I am going to take out the funnel and I'm going to wash the sides of the column using hexanes now you can see that the compound is nicely settled on the silica gel layer now I'm going to add some sand on the top of this mixture layer so for this I am taking some sand and I'm going to add it so why are you adding sand here oh we need to add the sand because the compound layer or the mixture layer will not be disturbed while we are adding the mobile phase during the experiment so once you add the sand you can tap it gently so that it forms the uniform layer on the top of your mixture layer so students from our TLC experiment we know that the yellow compound and the red compound can be separated with a t20 mixture of hexans and ethyl estate students you can see I made 50 ml of 80/20 mixture of hexane and ethyl acetate and I am going to use this as the mobile phase to elude the compounds in the column okay now I'm going to pour it very carefully so that I don't disturb the top layer so initially for few milliliters you can pour carefully then later on you can increase the speed and fill the column now I'm going to release the stopcock so that you will have some enough speed and the compounds will be eluted nicely you can see that the end of compound is actually coming first yes like the way that you saw it on the TLC ok yes and the red compound is at the bottom which you can see it here it's coming little slower wow it's beautiful you can see so you can say that the pattern is actually upside down compared to the top TLC because in TLC TLC is against the gravity whereas column is towards the gravity now at this point we need to take the test tubes in order to collect the compounds so you can see that slowly the ellow compound is coming however we are not collecting them in the test tube we will slowly collect it now you can see that the solution is becoming more yellow yes no I am collecting the first component which is the yellow combo yes and you can see the red compound is actually stuck on the top because it is moving slowly slowly students you can see that the yellow compound is actually moving much more faster compared to that of the red compound as one of my test tube is already getting filled I'll take a new test tube and put it here and I will collect more of the yellow compound and you can see that my mobile phase is getting used so I need to add more of the mobile phase which is the same 80/20 mixture of hexane and ethyl acetate and I'm going to add more of the mobile phase very carefully as you can see that the mostly yellow compound is already in neutered so again as my test tube is getting filled I will take hundreds of test tube and I will collect my yellow compound and students also make sure that the speed is uniform don't go it too much speed at the same time don't go too slow okay now you can see that all most of my low compound is eluted you can actually see a very nice space between the red one and lo1 there is actually the gap that's saying that the separation is very good you can see the same thing between the yellow one and the red one there is actually the gap that's saying that the separation is following same as the TLD NZ yes now students you can actually observe it carefully that most of the ellow compound is already eluted so now I can increase the polarity in order to elute my red compound okay I'm going to change the test tube again because you can actually see the difference between the two test tubes one is really yellow and the other one is almost white yes that is colorless that says that most of the yellow company is actually in now students you can see that the yellow compound is completely eluted now I'm going to increase the polarity to 50/50 hexanes Andy tell estate to elude my red compound okay so here you can see I have a mixture of 50/50 hexane and etl estate now I'm going to pour it here to elute my red compound again pour it carefully so that it doesn't disturb the top layer so I'll increase the speed a little bit I'm going to change the test tube now you can see that the low compound is completely eluted and the solution is colorless as I increase the polarity you can actually see that my red company is slowly started moving I can actually increase the speed of the column by opening the stopcock little bit more the red comp hole appears dark yellow because it's in dilute solution students now you can see that even the red compound is completely diluted okay now the column is finished I'm going to close the stopcock now students we have completely looted both the components of the mixture which we call them as Lu eights and I am showing them here the first three test tubes contain the yellow compound which is component and then fourth and fifth test tube do not contain any compound then the remaining test tubes contain the compound B which is the red compound but the compound of appears dark orange because it's in diluted form as shown on the screen from the structure we can see that silica gel is a polar adsorbent as yellow compound which is to nitro aniline moved faster compared to red that is methyl red it can be understood that to nitro aniline is less polar compared to methyl red the same thing was observed on TLC also now students the next job is to remove the solvent and use these compounds as pure compounds so students I hope you understood the working and basic principles of column chromatography if so could you summarize it yes so we have learned the principles of column chromatography it mainly consists of two phases one which is stationary phase which is silica gel here and the other is a mobile phase which is a mixture of it I acetate and hexanes here the compounds are purified based on adsorption and polarity yes sir we also learned that the polar compounds stick to the gel and move slowly whereas the nonpolar compounds are eluted faster we've also learned that TLC is performed on small-scale hence it is qualitative technique yes and column chromatography is performed on large scale hence it is quantitative technique well done students you have understood the concepts of column chromatography now it's your turn to use this technique on new compounds so when we meet next time we are going to work on another technique so till then goodbye [Music] [Music] you