Purpose: To identify plant pigments by separation and isolation of the pigments using thin layer paper chromatography.

Chromatography is a technique used to separate molecules on the basis of differences in size, shape, mass, charge, solubility and adsorption properties. The term chromatography is derived from Greek words Chroma-colour and Graphe-write. There are many types of chromatography: paper chromatography, column chromatography, thin layer chromatography and partition chromatography. These techniques involve the interaction between three components: the mixture to be separated, a solid phase and a solvent.

Retention factor or Rf value is applied in chromatography to make the technique more scientific than a mere analysis. The retention factor or Rf is defined as the distance travelled by the compound divided by the distance traveled by the solvent. The ratio of the distance traveled by a compound to that of the solvent front is known as the Rf value; unknown compounds may be identified by comparing their Rf's to the Rf's of known standards.

The different pigments in plants take a long time to separate, so you will need to set this up first and let it run while you are doing to other two experiments. The components that are attracted more to the paper will move very little, if at all.  The components that are more attracted to the mobile phase will travel with it, at different rates, depending on the level of attraction.  This component traveling process is called elution. 

Materials and Equipment- Chromatography paper, gel pens, and eluting solution, 600-mL beaker, pencil, ruler, plastic wrap, tape and paper towels.

1. Cut a strip of coffee filter (or filter paper). Draw a horizontal line with a pencil (not pen) about half an inch from the bottom.
2. Crush a spinach leaf and make a line of green pigment on the filter. Repeat this process until the line is fairly dark.
3.  Put about an inch of isopropanol (acetone, fingernail polish remover will work) in a beaker.
4. Tape the top of the coffee filter strip to a pencil and balance the pencil across the top of the beaker. See the image below for the set-up.
5.  It is very important that the bottom of the filter strip is in the isopropanol, but the green spot is not in the liquid. If the isopropanol touches the spot directly, the pigment will just dissolve away.
6.  Set the cup aside. The isopropanol will move up the filter paper slowly and deposit the pigment components along the way.

Analysis

1. Assign a band number for each pigment band - you should see greens, yellows, oranges..etc.

2. Explain how a crime lab could use paper chromatography to determine if lipstick found at a crime scene matched the lipstick of a suspect.

Safety-

1. Wear safety goggles at all times.
2. Use eluting solution only in the hood. 
3. Do not breathe fumes from the eluting solution. 
4. Be sure to handle only the dry part or the chromatogram when removing it from the beaker.
5. Wash hands thoroughly if the eluting solution touches your skin.