Separation of Chloroplast Pigments by Paper Chromatography

Place the beaker set-ups in a water bath and let boil until the pigment is extracted (when leaves are completely devoid of ______)

Do not boil under direct ______

Always boil in a water ______

Remove the leaves from the alcohol and spread them out in a ______ dish

Add just enough iodine solution to cover the ______

The presence of starch is indicated by a bluish black ______

Photosynthesis is the process whereby green plants convert the radiant energy of light into chemical energy that is then stored in ______ molecules.

For photosynthesis to occur, light, a green pigment and ______ dioxide must be present.

All living things are dependent on the products of photosynthesis for their energy, for the raw materials necessary for growth and repair and for the ______ necessary for life.

The process of photosynthesis takes place in ______.

Glucose, the main product of photosynthesis, can be converted into starch, a storage molecule that can be broken down to glucose again when the plant requires it, during ______ respiration.

At the end of the exercise, the student is expected to be able to measure the rate of photosynthesis as affected by some factors and detect the presence or absence of photosynthetic products in leaves, and extract and identify the pigments involved in ______.

Set the three test tubes in bright ______.

Chromatography is a method of separating closely related compounds by allowing the mixture of substances to diffuse through an absorbent called ______.

The different chloroplast pigments can be separated and observed as different color bands on the ______.

Chlorophyll pigments can be identified not just by their band color but also by the distance they travel through the ______.

The Rf (retention factor) value is a ratio that can be calculated and compared to known Rf standards to identify unknown ______.

Grind the leaves using a pestle to release the ______.

Add another 3 ml of ______ and continue crushing until the acetone becomes a very dark, highly concentrated extract.

Collect the extract by pouring the solution into a beaker covered with ______.

Fold the cheesecloth and squeeze the extract to release all the liquid into the beaker. Cover the beaker when ______.

Cut a strip of filter paper about 1 inch wide and long enough to reach the solvent at the bottom of the beaker that you will use as your chromatography “tank”. Cut the filter paper to the shape shown in Figure 1. This is referred to as a ______.

Using a pencil, draw a faint line 1 cm from the pointed tip. This marks your solvent’s origin. With the capillary tube/pipette, apply three to five drops of the pigment extract just above the ______ line.

Pour the prepared separating fluid consisting of a 1:9 mixture of acetone and petroleum ether into the chromatography tank (tall beaker) to a depth of a little more than 0.5 cm. ______

Carefully lower the extract-treated chromatogram into the tank, with the end immersed in the separating fluid but not reaching the level of the pigment spot. Secure the chromatogram with a ______.

Make sure that the chromatogram is suspended freely, with no sides touching the tank or other chromatograms. The solvent will move up the strip and carry the pigments with it. Observe the set-up around a 15-to-30-minute period as the separating fluid diffuses upward along the length of the chromatogram, passing through the spot and gradually separating the chloroplast pigments. Draw the separated pigments on your worksheet and label each color band by its corresponding ______.

Using a ruler, measure the distance from the origin line to the solvent front (visible as a thin, transparent line at the top of the chromatogram) and to the middle section of each color band (at its widest section). Calculate the ______ values for each pigment.

Fill in the table in your worksheet. Compare your ______ values with the known standards of each pigment for the specific solvent as follows: a.Carotene – 0.95 b.Phaeophytin – 0.83 c.Xanthophyll – 0.71 d.Chlorophyll a – 0.65 e.Chlorophyll b – 0.45 FORMATIVE LEARNING ACTIVITY: Labster: Refer to the Labster link in your canvas for the online simulations Simulation: Pigment Extraction: Use Photosynthesis to Produce Biofuel Simulation: Algae Pigment Analysis Adapted from: De Jesus J, Ragragio E. 2000.Laboratory Manual in General Botany.

Carefully lower the extract-treated chromatogram into the tank, with the end immersed in the separating fluid but not reaching the level of the pigment spot. Secure the chromatogram with a paper clip. Make sure that the chromatogram is suspended freely, with no sides touching the tank or other chromatograms. The solvent will move up the strip and carry the pigments with it. Observe the set-up around a 15-to-30-minute period as the separating fluid diffuses upward along the length of the chromatogram, passing through the spot and gradually separating the chloroplast pigments. Draw the separated pigments on your worksheet and label each color band by its corresponding pigment. Using a ruler, measure the distance from the origin line to the solvent front (visible as a thin, transparent line at the top of the chromatogram) and to the middle section of each color band (at its widest section). Calculate the Rf values for each pigment. Fill in the table in your worksheet. Compare your Rf values with the known standards of each pigment for the specific solvent as follows: a.Carotene – 0.95 b.Phaeophytin – 0.83 c.Xanthophyll – 0.71 d.Chlorophyll a – 0.65 e.Chlorophyll b – 0.45 FORMATIVE LEARNING ACTIVITY: Labster: Refer to the Labster link in your canvas for the online simulations Simulation: Pigment Extraction: Use Photosynthesis to Produce Biofuel Simulation: Algae Pigment Analysis Adapted from: De Jesus J, Ragragio E. 2000.Laboratory Manual in General Botany. This process is typically used in ______ analysis.

Carefully lower the extract-treated chromatogram into the tank, with the end immersed in the separating fluid but not reaching the level of the pigment spot. Secure the chromatogram with a paper clip. Make sure that the chromatogram is suspended freely, with no sides touching the tank or other chromatograms. The solvent will move up the strip and carry the pigments with it. Observe the set-up around a 15-to-30-minute period as the separating fluid diffuses upward along the length of the chromatogram, passing through the spot and gradually separating the chloroplast pigments. Draw the separated pigments on your worksheet and label each color band by its corresponding pigment. Using a ruler, measure the distance from the origin line to the solvent front (visible as a thin, transparent line at the top of the chromatogram) and to the middle section of each color band (at its widest section). Calculate the Rf values for each pigment. Fill in the table in your worksheet. Compare your Rf values with the known standards of each pigment for the specific solvent as follows: a.Carotene – 0.95 b.Phaeophytin – 0.83 c.Xanthophyll – 0.71 d.Chlorophyll a – 0.65 e.Chlorophyll b – 0.45 FORMATIVE LEARNING ACTIVITY: Labster: Refer to the Labster link in your canvas for the online simulations Simulation: Pigment Extraction: Use Photosynthesis to Produce Biofuel Simulation: Algae Pigment Analysis Adapted from: De Jesus J, Ragragio E. 2000.Laboratory Manual in General Botany. The Rf value represents the ratio of the distance traveled by the ______ to the distance traveled by the solvent front.