welcome biologist to part two of photosynthetic pigments in this video we're going to take a look at thin layer chromatography of photosynthetic pigments so usually what you do is you have your plant sample and the first thing you would do is to crush it using a pestle and water now what this helps to do is to break the cell wall and and some of the membranes to release those photosynthetic pigments that should be inside of the chloroplast so don't forget the chloroplast has that um membrane around it as well that double membrane which needs to be broken as well so once you release those photo synthetic pigments and you've got a kind of a paste in there you'd use a tubing to put a small dot of the mixture onto a pencil line that you've drawn on ethan layer chromatography now this space is called the stationary phase because nothing's moving and what we use here is silica gel now although gel implies that it might be more of a liquid solution actually the liquid is dried onto these pieces of if you like plastic and which is why it's still called silica gel even though it's dried silica gel so we would we must draw the line with a pencil so that the um line doesn't get dissolved within my solvent which is what is added next um but we must handle this thin layer chromatography um made with silica gel we must handle it with very care because amino acids on our hands might contaminate it and we don't want that to happen um now we've got to draw um this with a pencil line so that the solvent doesn't dissolve it like we mentioned before and what we've got to do as well is make sure that the pigment um dries in between applications because we need to make it as concentrated as possible and so what we do here is we would put a dot on the line allow it to dry and then put another dot on and i'll try now you normally do this about 10 times so the dots really really concentrated what you'd then do is you put the whole of the the nychromatogram into a solvent and this becomes a mobile phase now where the solvent will dissolve the photosynthetic pigments and take them up the chromatogram now again we've got to make sure here that the solvent must be below the pencil line otherwise our pigments will just go straight down into the solvent which we don't want we want them to separate out on the chromatogram so the smaller photosynthetic pigments will travel further at the chromatogram because there's less resistance between them and this and the sillier gel silica gel um so what you'd then do is you calculate your rf values now as you can see on this particular chromatogram you do get beautiful colours when you do this with photosynthetic pigments you can see where the different pigments end so what you would do is you do the distance moved by the compound over the distance moved by the solvent so that's the solvent front so you'll be doing the distance move by the compound so think so from here to here divided by for example here to hear the solvent from so the solvent up from the origin to the solvent front always goes on the bottom now you're usually measuring millimeters because it's more accurate than measuring in centimeters so for example that would be on the top and then you divide that by the solvent from measuring in millimeters now what you would then do is you can compare the rf value to known rf values so say for example you get an rf value of 0.42 you would know from this table of known standard rf values that that error value would be for example uh chlorophyll b and that pigment there would be chlorophyll b all right and so in an exam you do need to be prepared to be able to guesstimate and estimate what a pigment might be from a chromatogram you do need to be able to calculate rf values correctly as well so um those are the um correct pigments there on that i am chromatogram and that's pretty much all that you need to know on photosynthetic pigments and thin layer chromatography good luck with your exams and all the best you