[Music] paper [Music] chromatography chomatography is a technique used to separate molecules on the basis of differences in size shape Mass charge solubility and absorption properties in paper chromatography the mixture is spotted on the paper dried and the solvent is allowed to flow along the sheet by capillary attraction as the solvent slowly moves through the paper the different compounds of the mixture separate into different colored spots the paper is dried and the position of the different compounds is visualized different plant segments can be separated by using the technique of paper [Music] chromatography materials required fresh spinach leaves filter paper strips chromatography chamber MTAR and pesel ether acetone solvent measuring cylinder containing 5 ml acetone capillary tube thread stapler scissors spatula watch glass scale and a [Music] pencil [Music] procedure take a few freshly plucked green spinach leaves using scissors cut the spinach leaves into small pieces and let them fall into to the mortar take a measuring cylinder that contains 5 ml of acetone and pour it into the mortar grind the spinach leaves using the mortar and pesel place the extract into a watch glass using a spatula take a strip of filter paper having a narrow Notch at one end of the strip take a pencil and a scale and drw a horizontal line with the pencil about 2 to 3 cm away from the tip of the notch put a drop of the pigment extract in the middle of the line with the help of a capillary tube allow the drop to dry and repeat till four or five drops are placed on the paper take the chromatographic chamber and pour ether acetone solvent into it fold one end of the filter paper strip and stap it using a thread hang the filter paper strip into the chromatographic chamber the loading spot should remain about 1 cm above the solvent level leave the chromatographic chamber undisturbed for some time we can observe as the solvent moves through the paper it spreads the different pigments of the mixture in various distances when the solvent rises above 34 of the way up the strip remove the strip carefully and let it dry observations the dried chromatographic paper strip shows four distinct pigment bands different pigments can be identified by their colors calculations RF value of each pigment spot can be calculated by the equation RF is equal to distance traveled by the compound divided by distance traveled by the solvent measure the distance of each pigment band from the loading spot and also the distance traveled by the solvent calculate the RF value using the equation and record the values into a table [Music] result the topmost orange yellow band of pigments in the separation corresponds to kerene the yellowish band appearing below it indicates the zantho fils the third from the above dark green band represents chlorophyll a the lower most yellowish green band is that of Chlorophyll [Music] B precautions spinach leaves should be fresh and green the loading spot should be 2 to 3 cm away from the tip of the knot while hanging the strips in the chromatography chamber the loading spot should remain about 1 cm above the solvent level [Music]