PAPER CHROMATOGRAPHY - CHEMICAL AND MECHANICAL
 
VALERIE YOUNG                  KENWOOD ACADEMY 
                               5015 S. BLACKSTONE AVE
                               CHICAGO, IL 60615
                               1-312-536-8907

OBJECTIVES: 
      To determine all of the pigments present in the chloroplast of a green 
plant.  the student will prepare and read a chromatogram by removing, seperating, 
and identify the pigments by using a technique called chromatography.   

MATERIALS:  
      test tubes, test tube holder, filter paper (strip type) or chromatography 
paper, cork, thumbtack, green leaf (such as spinach, geranium,grass or the leaf off 
a tree), carrot leaves are most suitable.  They provide a good source of carotene.  
Glass pipette, tweezers, scissors, hotplate, small beaker (Pyrex), ethyl 
alcohol (use denatured.)  Acetone may be used to extract chlorophyl from the leaf.  
A glass rod, water and a solvent which consists of 45 ml petroleum ether, 5 ml 
acetone, and l ml benzene (this is enough solvent for 20 students) 

STRATEGIES:
      CHEMICAL PREPARING of LEAF PIGMENTS (May be prepared by one member of your 
class or the teacher.  From this preparation , enough pigment will be made 
available for the entire class.)  Each class member will then prepare his/her own 
chromatogram. 

 1.  Fill a 600 ml beaker 1/4 full of water.  Set this beaker on a hot plate. 
 2.  Bring the water to a boil.
 3.  Place entire package of spinach (if using frozen spinach) into the 
     boiling water.  Bring to a boil again..
 4.  After one minute, remove the spinach with tweezers and squeeze out all 
     excess water.  Then transfer the boiled spinace to a 400 ml beaker 
     containing 80 ml of ethyl alcohol.
 5.  Heat the beaker by placing it onto the hot plate.  Leave it on the 
     hot plate for only about 30 seconds or until the alcohol begins to bubble. 
     Allow the alcohol to cool.  Then reboil it several more times.     
 6.  Remove the beaker from the hot plate.  Squash the spinach with a glass  
     rod.  Reheat and squash until the alcohol solution becomes a dark green 
     color.  Enough pigment is now available for the entire class. 

PREPARING THE CHROMATOGRAM

Prepare a strip of filter paper chemically as follows:

 1.  Cut two small notches (one on each side of the strip) about 2 cm from 
     the bottom.
 2.  Use scissors to taper at the  bottom of the paper to a point.
 3.  Attach the filter paer strip to a cork using a thumbtack and position 
     the strip so that when inserted into a test tube, the filter paper tip just
     touches the bottom.  Adjust the height by moving the strip either up or 
     down on the cork.(The filter paper strip should not touch the sides of the 
     test tube.  Trim if necessary.)
 4.  Dip the fine end of a tiny glass pipette into the pigment solution 
     prepared in the first section.  
2
 5.  Touch the pipette to the filter paper between the notches. Allow the 
     pigment solution to flow onto the paper.
 6.  Allow the spot to dry (about thirty seconds).  Then add more pigment 
     solution to the same spot.  Make 20 applications of the solution.  Allow 
     time for drying between applications.

Prepare a strip of filter paper mechanically as follows:  

 1.  DO NOT CUT NOTCHES ON THIS FILTER PAPER STRIP but taper the bottom as 
     in the chemically prepared paper.
 2.  About 3 cm from the bottom take a leaf you have previously collected 
     and a coin (preferrably a dime)
 3.  Place leaf on the filter paper and rub the dime across the leaf about 
     six or seven times removing the pigment onto the paper.


SEPERATING THE PIGMENTS:

CAUTION:  SOLVENT IS HIGHLY FLAMMABLE.  Before proceeding, all flames in the 
laboratory must be extinguished.  Make sure the room is well-ventilated.  
Do not inhale fumes.

 l.  Add solvent to a height of l cm in the test tube.
 2.  Place the filter paper strip into the tube. It is important that the 
     pointed tip just touch the solvent.
 3.  DO NOT move or shake the tube for at least 15 minutes.  Remove the 
     paper chromatogram from the test tube when the level of solvent almost 
     reaches the the top of the paper strip.
 4.  Examine the chromatogram for the presence of different bands of color.  
     Each color band is a different pigment.  You will find the following 
     pigments:   CAROTENE - at the top of the paper - ORANGE
            XANTHOPHYLL I - in the middle of the paper - YELLOW
            XANTHOPHYLL II - in the middle of the paper - YELLOW (may be 
              only one yellow band)
            CHLOROPHYLL a - toward bottom - BRIGHT GREEN
            CHLOROPHYLL B - lowest on paper - KHAKI GREEN