hello everybody my name is Iman welcome back to my YouTube channel today we're continuing our lecture on separations and purifications we made it to objective three which is all about chromatography now chromatography is another tool that uses physical and chemical properties to separate and identify compounds from a complex mixture now in all forms of chromatography that we're going to discuss today the concept is really identical the more similar a compound is to its surroundings whether by polarity charge or other characteristics the more it will stick to and move slowly through its surroundings all right kind of like how if you encounter people that are very similar to you you're going to get comfortable you're going to stay around them for longer all right just a little analogy very not scientific all right but just to help you think about it the more similar a compound is to its surroundings the more it will stick to and move slowly through its surroundings now the process really begins by placing this sample onto Some solid medium that we call the stationary phase or the absorbit now we then run the mobile phase which is usually a liquid sometimes a gas through that stationary phase this will essentially displace or elute the sample and carry it through the stationary phase depending on the characteristics of the substance in the sample and the polarity of the mobile phase it will adhere to the stationary phase with different strengths and that's going to cause the different substances to migrate at different speeds and this is called partitioning and it represents an equilibrium between the two phases different compounds are going to have different partitioning coefficients and that means they're going to elute at different rates this results in separation within the stationary phase and that is going to allow us to isolate each substance we're interested in individually now there are many different media that can be used as the stationary phase each one exploiting different properties that are going to allow us to separate our desired compound we're going to talk about different kinds of chromatography here today and we're starting off with thin layer and paper chromatography thin layer and paper chromatography are extremely similar techniques the only thing that they differ in is the medium that's used for the stationary phase so for thin layer all right for thin layer chromatography a thin layer of silica gel all right silica gel or alumina or alumina all right is used all right so for thin layer chromatography a thin layer of silica gel or alumina adherent to an inert carrier sheet is going to be used for paper chromatography as the name suggest the medium used is paper which is usually composed of cellulose now for these techniques the sample that we want to separate is going to be placed directly on the absorbent itself so if you have a silica kind of sheet here all right a thin layer of silica gel on a sheet all right you can go ahead and you can place your sample here all right so we place directly on the sheet our sample this is called spotting because we app place a small well-defined spot of the sample direct directly onto the silica or paper plate the plate is then dissolved which involves placing the absorbent upright in a developing chamber so what we're going to do is we're going to take this we're going to place it in a chamber that has a little bit of solvent all right now you're going to place that thin layer in here all right and make sure that where you dotted your sample is above the solvent all right so this at the bottom of this chamber this jar is a shallow pool of solvent we're going to call it the uent and this spots on the sample they have to be above the level of the solvent if they're not then what's going to happen is it's that sample is going to dissolve into the pool of salvent rather than running up this plate all right rather than running up the plate now when you set it up correctly the solvent will slowly start to creep up all right it's going to slowly start to creep up the plate by capillary action and it's going to carry the various compounds in the sample with it at varying rates now when the solvent front nears the top of the plate then you're going to remove it all right the plate is removed from the chamber and it's going to be allowed to dry so at some point it's going to reach a point right here and you're going to go ahead pop it out and then you're going to look at it all right so it's going to look something like this for example now as mentioned before TLC thin layer chromatography it's done with silica gel that's going to be polar and hydrop hydrophilic the mobile phase on the other hand is going to usually be an organic solvent of weak to moderate polarity so it doesn't bind to the gel now because of this non-polar compounds dissolve in the organic solvent and they move quickly as the solvent moves up the plate whereas the more polar molecules are going to stick to the gel and so what you can say is that the more non-polar the sample is the further up the plate it will move now you can also have a reverse phase chromatography which is just the exact opposite so in that technique the stationary phased used is non-polar so polar molecules are going to move up the plate quickly while the non-polar molecules are going to stick more tightly to the stationary phase now you take this out of the chamber you're looking at it the spots of individual compounds are usually white which makes them sometimes difficult or impossible to see on the white paper or TLC plate and so to get around this problem you can take this developed plate and you can place it under ultraviolet light which will is going to show you all right any compounds that are ultraviolet sensitive you can also use other things to stain the spots as well all right you're going to want to use things that are not going to destroy the compounds iodine is a good example now when TLC is performed compounds are generally identified using using this RF Factor all right it's used uh it's going to be determined using this RF Factor so this factor is relatively constant for a particular compound in a solvent and it's calculated using this equation it's the distance that the spot has moved over the distance the solvent has moved so if we're looking at this TLC plate right here all right this is going to be the distance all right the total distance that the um solvent has traveled all right we're going to note that as B then you can measure how much each of these points have traveled and you can write this ratio this is going to give you the RF Factor now because its value is relatively constant the RF value can be used to identify unknown compounds all right now this technique TLC paper chromatography usually used on small scale to identify unknown compounds sometimes it's used on a larger scale as a means of purification um this technique is usually called preparative TLC we don't have to get into the details of that for the MCAT but there we have it thin layer and paper chromatography now the next thing we want to talk about is column chromatography the principle behind column chromatography is the same as thin layer chromatography but there are a few differences First Column chromatography uses an entire column all right it uses entire column filled with silica or aluminum beads as an absorbent and that's going to allow for much greater separation in addition the way that thin layer chromatography works is using capillary action to move the solvent up the plate but column chromatography uses gravity to move the solvent and compounds down the column now you can also speed up this process you can force the solvent went through the column using gas pressure which is a technique called flash column chromatography now in column chromatography the solvent polarity it can be changed to help elute the desired compound and eventually the solvent drips out at the end of the colum uh at the end of the column and then different fractions all right that leave the column can be collected over time and each fraction that you collect over time is going to contain different compounds and so after collection the the solvent can be evaporated and then all you have left behind are your compounds of Interest all right now column chromatography is really really useful in Biochemistry because it can be used to separate and collect macro molecules like proteins or nucleic acids there are a couple of different techniques um and we're going to talk about them that fall under this category of column chromatography three specifically that we want to just briefly Define and be familiar with first one is Ion exchange chromatography here in this technique the beads in the column are going to be coated are they are going to be coated with charged substances so that they attract or bind compounds that have opposite charge so for instance a positively charged compound will attract and hold a negatively charged backbone of DNA or protein as it passes through the C that's going to either increase its retention time or retain it completely and so you can get everything else separated and after all the other compounds have moved through the column and you've separated everything else you want away from your DNA of interest for example then you can use a salt gradient to elute the charged compounds that were stuck to the column this whole time and that way you get to separate everything else you don't want from what you do want using ion exchange chome chromatography you also have size exclusion chromatography here the beads that are used in the column contain tiny pores of varying sizes and these tiny pores allow small compounds to enter the beads and and and as a consequence it's going to slow them down large compounds are not going to fit into the pores so they're actually going to move around them and travel through the column faster I know that's kind of counterintuitive so it's important to remember remember that in this type of chromatography the small compounds are actually slowed down and retained longer whereas the larger compounds make it through faster all right and the sizes of the pores they can be varied so that molecules with different molecular weights can be fractionated all right so that is size exclusion chromatography lastly we can talk about Affinity chromatography in Affinity chromatography a protein of interest is going to be Bound by creating a column and packing it with high and packing it with material that has high affinity for that protein and this can be accomplished by coding beads all right cating beads with receptors that are going to bind to a protein of Interest or a specific antibody of Interest Etc all right and so you pack it with materials that have these receptors that are going to bind to your protein of Interest and so that protein is going to be retained in the column and that way you can separate everything else out you don't want and then afterwards once the protein is uh once everything else is removed and it's the time to get the protein out of the column you can use a different kind of solvent to remove the bound proteins from these receptors and collect it at the bottom of the column so those are three different types of Colum column chromatography ion exchange size exclusion and Affinity now that with that being said we can move into another kind of chromatography called gas chromatography now this can get complicated but we don't know need to know too much about this for the mcast so we're going to keep it basic brief and to the point gas chromatography this is another method that can be used for qualitative separation all right gas chromat graphy is really similar to the other types of chromatography we've talked about but the main conceptual difference is that the uent is a gas instead of a liquid now the absorbent is going to be crushed metal or polymer that's inside of this 30t column about 30t column the column is coiled and kept inside of an oven to control its temperature the mixture is then injected all right it's going to be injected into all right it's going to be injected into the column and vaporized and the g the gaseous compounds are going to travel through the column at different rates because they adhere to the absorbent in the column to different degrees and that means they will separate in Space by the time they reach the end of the column now the injected compounds have to be volatile so that means they have to have a low melting uh point they have to be or or or have to be vaporizable liquids now the compounds are registered by a detector at the end here that will record them as a peak on a chart now it's really common to separate molecules using gas chromatography and then take it and inject the pure molecules into a mass spectrometer for molecular weight determination so Mass spectroscopy it's going to involve the ionization and fragmentation of compounds and then those fragments are run through a magnetic field which will separate them by has to charge ratio the total molecular weight can then be determined or the relative concentration of the different fragments can be calculated and then compared against some reference values to identify the compound last but not least we're going to talk about high performance liquid chromatography this is also can get really complicated but we're going to keep it brief and to the point high performance liquid chromatography um was previously called high pressure liquid chromatography and as the name suggests the uent is a liquid and it travels through a column of a defined composition there are a variety of stationary phases that can be chosen depending on the target molecule and the quantity of material that needs to be purified now this is really similar to column chromatography because the various compounds in solution are going to react differently with the absorbent material in the past very high pressures were also used but recent advances allow for much much lower pressures and so hence the change in that name now in high in hlc a small sample is going to be injected into the column and separation occurs as it flows through that uh through that column the compounds then pass through a detector and are collected as the solvent flows out of the end of the apparatus so this interface is really similar to that used for gas chromatography because the entire process is computerized but it just uses liquid Under Pressure instead of gas all right so with that we have covered everything we need to know about chromatography all forms of chromatography use two phases to separate compounds based on physical or chemical properties all right we learned the stationary phase or the absorbent is usually a polar solid the mobile phase runs through the stationary phase and is usually a liquid or a gas we talked about thin layer and paper chromatography we've talked about column chromatography and we talked about three variations of that ion exchange size exclusion and Affinity chromatography and then we ended the chapter with a discussion on gas chromatography and high performance liquid chromatography all right with that we've ended our lecture on the last chapter for mcow organic chemistry I hope this was helpful let me know if you have any questions comments concerns down below other than that good luck happy studying and have a beautiful beautiful day future doctors