Separation of Photosynthetic Pigments by Chromatography

How you can separate leaf pigments by a process known as chromatography?

 In this example I'm going to use spinach leaves

so the first step is for you to obtain your plant that contains the pigments you wish to separate break up one of the leaves and de little pieces and place it in a mortar add a few drops of acetone and a little bit of sand as this helps with grinding up the leaves use the pestle mortar to grind up the leaves and to release the pigments if it helps here  you can add a few more drops of acetone but ultimately the objective is to obtain a concentrated pigment in a liquid at the bottom of the mortar.

Chromatography Paper

after you've done this then you need to prepare your chromatography paper now this does actually work best with thin layer chromatography paper but you can also use regular chromatography paper to prepare you're going to obtain a pencil and it is important that it's a pencil not a pen because a pen contains other pigments use the pencil to draw a line on the chromatography paper that's about 2 centimeters up from the bottom of the paper you can see that here

Spotting

at this point you're going to place a spot of concentrated pigment on the line now this can be done in several different ways here I'm using a very small narrow plastic pipette it can also be done with a capillary tube and sometimes people do it with a very fine paintbrush but the most important part is to make that spot as concentrated as possible so in between spotting you can dry it with a hair dryer to make it more concentrated and then spot on top and you're going to keep doing this until you have a very dark green concentrated spot of pigment on your line the next step is to prepare your

Preparing the solvent

running solvent so you're going to take the container in which you will place your chromatogram and you're going to pour your running solvent into the container ensuring that it does not go above where the pencil line is that on your paper so in our case that would be not above 2 centimeters from the bottom of the container now there are a variety of different running solvents that people tend to use one of the most frequent is nine parts petroleum ether to one part acetone but it is possible that your teacher might give you a different running solvent to use in any case you would perform the same actions here but just with the solvent that you are provided with once

Chromatography

you've prepared your container you place your chromatography paper gently into it and allow the solvent to begin to move up the paper carrying the different pigments with it it's important to keep an eye on the solvent front because the pigments will stop at different distances relative to how far the solvent front goes one fun thing to do

Time lapse

here is to record it as a time-lapse video in these videos you can see how the solvent front moves up and the different pigments stop at different distances along the chromatography paper and this is going to be relevant in our calculations soon as I said the most important thing is to keep an eye on where the solvent front is and when the solvent front stops moving and you've observed that for a period of time it's not moving anymore you need to mark where the solvent front went to with a pencil immediately once you've marked where the solvent from went to then you need to measure the distance it traveled from the point of origin which is the original pencil line that you drew on the chromatography paper this is known as the distance traveled or moved by the solvent front next you want to mark the front edge of each pigment so that's each color and you want to measure how far each pigment or each color moved from the point of origin the original pencil line that you drew.

Calculating RF values

in order to identify the pigment you first need to calculate the RF value for each pigment

 that you found on your chromatogram to do this for each one you want to take the distance move by the pigment presumably in millimeters and divide this by the distance moved by the solvent front also in millimeters once you've calculated the RF values for each pigment that you observe on your chromatogram you can compare it against known published RF values in order to identify the pigments on your chromatogram and therefore the pigments in your leaf for example the published RF value for carotene tends to be between zero point eight nine and zero point nine eight so if you obtain a number within this range and it tends to be a yellow orange color it's likely to be the pigment carotene you can see it here at the top of the chromatogram very close to the solvent front the published RF values are easy to research and look up and there are many tables available for you to compare against and identify your pigments but for example this website also offers additional resources including the written protocol to help support you when carrying out this practical in the lab.

for video click on below link thanks!!

https://youtu.be/W56RHxu2Hpc