INTRODUCTION

 Photosynthesis is the process that plants use to convert CO₂ into carbohydrates (sugar).  These carbohydrates are stored (as starch) and are used by the plant for food.  The basic formula for photosynthesis is:

 There are 3 photosynthetic pathways in plants known as C₃, C₄, and CAM.  C₃ plants are the most common.  These plants attach CO₂ to the 5 carbon sugar RUBP (ribulose bisphosphate) with the assistance of the enzyme RUBP carboxylase which will then be converted into sugar.  C₄ plants, like corn, first convert CO₂ into malate and then malate is converted to sugar by RUBP carboxylase.  All this takes place during the day.  CAM plants (e.g. cactus) first convert CO₂ into malic acid.  This happens at night.  During the day CO₂ is released from malic acid, and converted into sugar with the aid of RUBP carboxylase.

EQUIPMENT
    Electronic balance, pH meter

SUPPLIES
     2 CAM plants- succulents (1 in sun for 24 hours and 1 in dark for 24 hours), scissors, mortar and pestle, sand, distilled H20, 10 mL pipette, pipette pump, wash bottle (distilled water), beaker to wash pH meter electrode, 30 mL beakers, stirring rod, grease pencil.

PURPOSE
    In this lab we will determine which of the CAM plants tested was left in the dark for 24 hours or left in the sunlight for 24 hours.

PROCEDURE
    Extraction and pH measurement

1. Remove leaf from unknown CAM plant A.

2. Wash leaf, blot dry with a paper towel.

3. Weigh approximately 0.5 gm of leaf (use scissors to cut pieces of leaf) to 0.01g.

4. Grind in a mortar and pestle with sand and 3.0 ml distilled water.

5. When the leaf material is a homogenous mixture,  put into a 30 mL beaker, add 10.0 mLs of distilled water     and mix thoroughly with a stirring rod.

6. Determine pH of solution with pH meter.

7. Repeat procedure 1-6 with unknown CAM plant B

1. pH of CAM plant A                                          ________________

2. pH of CAM plant B                                          ________________

1. Which plant was left in the dark for 24 hours?

2. Which plant was left in the light for 24 hours?

3. Explain how you determined the answers to questions 1 and 2.

4. Why do you think it would be advantageous for a plant to take in CO₂ only at night rather than during the day?

5. What environment might have many CAM species?

 

INTRODUCTION
    Chromatography is a technique used to separate mixtures of compounds.  In both thin layer chromatography (TLC) and paper chromatography a spot of mixture is put onto a chromatography plate or paper.  The end of the plate or paper is put into a solvent.  As the solvent creeps up the paper and past the spotted mixture of pigments, some of the pigments dissolve in the solvent more quickly than others.  After a period of time the different pigments in the mixture end up spread out between the original spot and the point the solvent reaches.

PURPOSE
    To use chromatography to separate and compare the photosynthetic pigments found in algae and green plants that grow in different light conditions.

EQUIPMENT
    UV Illuminator

SUPPLIES
    Safety goggles, centrifuge, mortar and pestle, acetone:water (80:20), capillary pipettes (25 µL), TLC plate (cellulose on plastic), 600 mL glass beakers, aluminum foil, solvent (petroleum ether:acetone  92:8), fine grain sand, plant material, centrifuge tubes.

PURPOSE
    The purpose of this exercise is to extract pigments from selected plants, separate the pigments with thin layer chromatography, and compare the results for different types of plants.  Your teacher will explain the different types of plants you will be using.

PROCEDURE
    1. Weigh out approximately 0.5 g of plant material on the analytical balance.  Use scissors to cut up your sample.

    2. Grind the plant material in 4 mL 80% acetone which you have pipetted from the large beaker.

    3. Pour extract into numbered centrifuge tube.

    4. Match with another tube of similar volume.

    5. Centrifuge for 5 minutes, then decant the clear green liquid into a labeled glass vial.

    6. Carefully mark your TLC plate as instructed by your teacher. (See Figure 1).

Figure 1     7. Following the teacher's demonstration, carefully spot 50 µL of your plant extract onto your marked TLC plate, using the capillary pipettes.   This will require 30-50 droplets applied on the same spot.   Remember that you want small, dark spots for best results.

    8. Obtain a different plant extract from another lab group and using a new capillary pipette,  spot 50 µL about 1 cm from the first sample.   Repeat with additional extracts.   Allow to air dry.

    9. Carefully insert your TLC plate into the chromatography chamber.   Solvent should not touch the spots.   Cover the beaker with foil.   See Figure 2.

Figure 2

   10.  Remove the TLC plate when the solvent front reaches within one cm. of the top of your plate (about 10 min).   Circle pigment spots on the plastic side of the plate.

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Plants- Using Figure 3, draw a chromatogram of each plant using colored pencils .  Compare the pigments found for different types of plants.

Figure 3