Now, let’s separate the pigments from fresh spinach leaves using paper chromatography.
Take a few freshly plucked green spinach leaves.
Using scissors, cut the spinach leaves into small pieces and let them fall into the mortar.
Take a measuring cylinder that contains 5ml of acetone and pour it into the mortar.
Grind the spinach leaves using the mortar and pestle.
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 draw a horizontal line with a pencil about 2-3 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 in it.
Fold one end of the filter paper strip and staple it.
Using a thread, hang the filter paper strip in 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 to various distances.
When the solvent rises about 3/4th up the strip, remove the strip carefully and let it dry.
You can select the type of leaf extract using the drop down list, ‘Select the plant pigment extract’.
Now, select the type of solvent using ‘Select the solvent’ drop down list.
Click on the ‘start’ button to start the experiment.
We can observe that the different pigments move through the filter paper to various distances.
Click on the test tube to measure the R_f value. There appears a tooltip to identify the band.
Select the ‘show scale’ check box to measure the distance travelled by the pigment and solvent.
Select the ‘show chart’ check box which displays a chart of predefined values of different leaf pigments.
Check the calculated Rf value that matches the predefined values in the chart.
Now select the appropriate pigment band from ‘Select the pigment identified’ drop down list.
To repeat the experiment, click on the ‘Reset’ button.
The dried chromatographic paper strip shows four distinct paper bands. Different pigments can be identified by their colours.
R_f Value of the each pigment spot can be calculated by the equation;
R_f=(Distance travelled by the compound)/(Distance travelled by the solvent)
Measure the distance of each pigment band from the loading spot and also the distance travelled by the solvent. Calculate the Rf value using the equation and record the values in the table.
The topmost orange yellow band of pigments in the separation corresponds to carotene. The yellowish band appearing below it indicates the xanthophylls. The third from above dark green band represents chlorophyll a. The lowermost yellowish green band is that of chlorophyll b.
