BIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017

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Biology Chapter 2: Chemistry of Life

Biology Plant Pigments and Chromatography Lab

Mrs. Douma 2017

Plant Pigments and Chromatography




Background Information


Photosynthesis begins when light is absorbed by pigments in the plant cell. One technique for separating and identifying these pigments is paper chromatography. In paper chromatography, solvent moves up the paper by capillary action (adhesion) carrying with it dissolved solutes – in this case plant pigments. The pigments are carried along at different rates because they are different sizes, not equally soluble in the solvent and are attracted in different degrees to the paper (adhesion and hydrogen bonding).


Many green leaves contain pigment colors that are not seen until autumn because they are hidden by the chlorophyll. A few plants have leaves that are red, orange, or yellow all year long.


Beta carotene, the most abundant carotene in plants, is carried along near the solvent front because it is very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose (non-polar). Xanthophyll is found further from the solvent front because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the cellulose. Chlorophylls contain oxygen and nitrogen and are bound more tightly to the paper than are the other pigments (so they will move more slowly.)


Chlorophyll a is the primary photosynthetic pigment in plants. Chlorophyll b and the carotenoids (xanthophylls and carotene) are also present. Carotenoids also protect the photosynthetic system from the damaging effects of ultraviolet light



Objectives

  1. Separate plant pigments using chromatography

  2. Observe and identify the different pigments found in a leaf.

  3. Understand how chromatography separates two or more compounds that are initially present in a mixture

  4. Calculate the Rf value of a pigment



Procedure

  1. Obtain a graduated cylinder that has 1 cm of solvent in the bottom. The cylinder should stay stoppered because the solvent is very volatile. Avoid breathing into the cylinder.

  2. Cut a piece of filter paper that is just long enough to reach the solvent. Cut one end of the paper into a point and then draw a light pencil line 1.5 cm above the point.

  3. Use a coin to extract the pigments from the spinach leaf cells. Place a small section of leaf on the top of the pencil line. Use the edge of the coin to crush the cells. Be sure that the pigment line is on top of the pencil line. Make two or three passes, using a different part of the leaf each time.

  4. Place the chromatography paper in the cylinder so that the pointed end is immersed in the solvent. Do NOT allow the pigment to be in the solvent.

  5. Stopper the cylinder. When the solvent is about 1 cm from the top of the paper, remove the paper and immediately mark the location of the solvent front before it evaporates.

  6. Mark the bottom of each pigment band with a light pencil.

  7. Measure the distance each pigment migrated from the bottom of the pigment origin (pencil line) to the bottom of the separated pigment band. Record the distance in mm that each pigment moved.



BIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017
BIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017


BIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017

BIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017










ABIOLOGY PLANT PIGMENTS AND CHROMATOGRAPHY LAB MRS DOUMA 2017 nalysis


  1. What does a small Rf value indicate about the characteristics of moving molecules? Hint – Read the background information









  1. Which are more soluble in the chromatography solvent, xanthophylls or chlorophyll a?





  1. Would you expect the Rf value of a pigment to change if we altered the composition of the solvent? Why or why not?







  1. If yellow xanthophylls were present in the extract (crushed leaf) why did the extract appear green?








  1. Is it possible to have an Rf value greater than 1? Why or why not?






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