 In this video, we will show you how to perform a column. Before you perform a column, you will have to have done a TLC of your product and have a good separation on TLC. If you do not know how to perform a TLC, watch the video about tin layer chromatography. If you want to perform a column, you will need the following pieces of equipment. First of all, you will need a column. In this column, you will have to fill with a couple of ingredients, some cotton, sand. In this case, we will use silica gel, but you can also do alox columns. You will need to run the column with a solvent. You will have to decide which solvent you have to use, or it's in your manual. In this case, solvents come in glass bottles. We use pentane and diethyl ether. You will need to mix your silica and your solvent. For that, it's handy to use a big beaker. To accurately determine the mixture of your solvents, you can use a measuring cylinder. To add solids to the column, it's handy to have a big funnel. We want to put some pressure on our column. For that, you can use a mill adapter attached to a small balloon which you can press and then you will have pressure on your column. To attach the balloon to your column, use a fitting clamp. And to collect the fractions at the end of your column, you will need a test tube rack filled with test tubes. And of course, you will need your crude product, which you want to call. We already placed the column in our fumeut in the shot and we placed it with two clamps, one on the top and one somewhere in the middle. Make sure these clamps hold the column tightly so it can't go anywhere and it is not tilted to any of the sides, backwards or forwards to have a level flow through your column. At the bottom of the column, we placed a tiny bit of cotton pressed down with a long rod and this is to prevent any of the sand or the silica that we are going to place in here or any other solid face that you are going to place in here from leaking out the bottom of your column. And above the cotton, we have a layer of sand to have a nice equal bottom layer of your column. Now that we have prepared this, we can continue to prepare the rest of the column but before doing that, make sure that you check by TLC that your product that you want to isolate is at an RF value of around 0.3 in the element that you want to use. In other case, it's the red spot or the top spot for those that are color blind and the blue spot is at the bottom and that is our impurity that we don't want. Now we will continue with making our column. By first putting a layer of element at the bottom to prevent the sand from getting disturbed and after we placed the element in, we can add our silica and silica is a very free flowing powder and you can load it into your column in two ways. You can either load it like this in a dry form but that won't give a good packing and it will cause a lot of dust and this dust is quite harmful to your lungs. The other option would be and the better option to use is to make a slurry of your silica using the elements that you want to use and then pour that slurry into the column and then let the liquid drain to bit the backer piloting as we will show you now. We prepared 500 ml of our solution that we're going to use, our elements but Bosn always make sure that there's enough solvent around in the lab that you can actually finish your column before starting your column. Use a small pipette to fill the column with a layer of elements and make sure that the tap at the bottom of your column is closed while doing this because if it's not closed it will run dry. When you have a layer of about a centimeter that should be enough and we can now load the silica into the column. So we have our silica and their elements and you just mix this until you get a slurry-like consistency. To help mixing you can use a spatula or the backside of a pipette or just swirl until it's properly mixed. So now that our slurry is properly mixed we can pour it into our column and for that we're going to use a big funnel so that it will not stick to the sides of our glass preferably and be careful when pouring this but still pour fast enough that you actually pour in the silica and sometimes you will have to refill the beaker with a bit more elements to actually get the silica that you wanted to go in to go in. As you can see the silica is slowly settling down in our liquid but there's still a bit of silica on the top which you can rinse away with a small pipette and some of the elements. As you see the settling will take quite some time and we can improve the settling time by opening the tap and applying some pressure but make sure that when you're doing that the silica never runs dry of solution. As you can see we let the liquid drain until just below the layer of the silica and now we'll place our compound on top of the column. We dissolved our crude mixture in a minimum amount of our element and we will place a tin layer depending on how much you have of course on top of the column. Try to not disturb the top of your silica layer while doing this. Now that we placed our compound dissolved in a minimum amount of element on top of our column we can open it and let it run into the silica and while it's almost in you can rinse the sides of the column with a tiny bit of element and also let that extra bit of element flow into your column. Now it's in the column, close the column and cover the top with a small layer of sand to prevent any compound going back into the element that you will place on top of your column. To do this use a funnel for solids and the sand and a small layer is enough. Now you can first carefully fill the top layer with element and when there's a small layer of element you can pour in the rest of the element. Now that there's a small layer you can pour in more element and open the tap at the bottom. The first 60% of liquid volume of our column will be just pure element because it was just mixed with pure silica. So we can collect that in a large earlamire and reuse that to flow through it again. After around 60% of this volume, so that is about this which is a similar amount that was on top of our column now, is run through we will start collecting our fractions. But now we will just push through 60% of volume. Make sure you always top off the solvent before the sand runs dry. Now that around 60% of our volume has run through we will start collecting fractions. In our demonstration that you see here we have very coloured fractions and it's easy to see where they are. But in most experiments you're trying to separate a colours compound from another colours compound. We can fill these test tubes for whatever volume you like but make sure that you don't overfill them and the liquid flows out. As soon as you are around halfway to three quarters full exchange it for the next one and place it in order in your reaction in your test tube rack or you could number your test tubes. In this case we can clearly see that there is a very yellowish tint here which is probably one of the fractions. Then there is this bright red fraction which we could also observe on TLC and then there is this blue fraction which we also could observe on the TLC and we would like to have pure red compound at the end of this column. In this case we are doing flash chromatography so we are applying pressure on top of our column. This is because when you are doing this fast so you want to do this in one go there is no chance or lower chance of diffusing of these layers into each other so by continuously going on and collecting our fractions not stopping in between we will have the best separation possible as we will now demonstrate in the following montage. When you think you are done collecting fractions make sure you leave a tiny layer of element on top of your column and close the tab. So here you can see the fraction that is collected and you can clearly see that the color changes from light yellow all the way to darker red and then now in this way more dark red at some point the dark red goes through colorless here and then the colorless goes from light blue to dark blue and then all the way to light blue again and the last fraction I collected was almost colorless there is a tiny amount left in the column but for now that is okay. Now we just have to identify from these fractions which contain pure compound and you can do this by running TLCs of every fraction or every other fraction to see which fraction is pure and then when you identified all your pure fractions combine them into a big flask use the rotary evaporator to remove the solvent and identify what you isolated by using any of the analytical techniques When you are sure that you have your product isolated in your flask you can empty the column by opening the tab and pushing out the liquid using the balloon make sure that you collect this liquid in Erlemire for the one case that you actually did not isolate your product yet when the column is completely dry or at least dry enough you can hang it upside down in your fume hood over a beaker and leave it there overnight and then the next day the silica will have dropped out of your column and you can throw it away in the solid waste bin and clean the rest of the column with some acetone and ethanol at the waste fume hood this is how you perform a column