Paper Chromatography of Pigments in a Spinach Leaf

What is the purpose of this experiment?

The purpose is to find the individual pigments that give spinach its dark green color, and we are using a separation method called paper chromatography to help isolate each pigment.

What do you need to make sure of during the experiment?

Make sure that the pigment spot is not immersed in the solvent

How is paper chromatography done?

Paper chromatography separates a pigment mixture by placing a spot of it on paper above a solvent, which then travels up the paper and carries different components at different speeds based on their solubility.

Stationary Phase

Paper Strip

Mobile Phase

It occurs at the end of the paper strip containing the pigment mixture spot. It is then placed in a small amount of the solvent in the beaker.

What pigments will you be identifying in this experiment?

• Chlorophyll a: Blueish green color

• Chlorophyll b: Yellowish green color

• Carotene: Light yellow orange color

• Xanthophyll: yellow to grayish color

• Anthocyanin: Red 

Why is anhydrous magnesium sulfate being used?

Helps remove the water from the spinach leaves

How long do we let the spinach pulp mixture stand after we are done grinding it down with a pesto and mortar?

10 minutes

How much should the solvent cover the bottom of the chamber?

0.5 cm in depth

How long should we draw the base line for our chromatography?

1 cm above the base of the paper. This is where we will deposit our sppinach pigments. Mark a dot on the line and make sure to label the letter “e” for the leaf extract.

What is the chromatogrpahy solvent mixture made out of?

Acetone and petroleum ether

How do we make a concentrated dot of extract?

Fill the capillary tube with leaf extract, then repeatedly touch it to the center dot on the TLC plate (letting it dry between applications) until you have a small, concentrated, visible spot. This process must be repeated several times.

What do we need to be aware of when we are inserting the paper into the chamber?

The bottom of the paper shall be immersed in the solvent, but make sure that START line (Base line) isn’t immersed.

How long do we allow the paper to develop and the pigments to separate?

10 minutes or until the solvent has reached about 1 cm from the top of the paper

What is the solvent front?

It is the highest level the solvent reaches as it moves up the TLC paper.