Paper Chromatography Lab Report


Introduction

Background Information
The word chromatography comes from the Greek and literally means color
writing. The technique was first described by the Russian botanist Mikhail Tswett
in 1903. He dissolved the pigments of green leaves in a mixture of petroleum
ether and alcohol, poured some of the resulting deep green solution onto
powdered chalk packed in a vertical glass tube, let it soak in, and then added
more solvent. The colored constituents of the leaves were washed down the
tube. The carotenes, which are weakly absorbed by the chalk, moved ahead,
while the chlorophylls, which are strongly absorbed, moved more slowly. Soon
the tube showed bands of color-orange, green, and yellow-separated by white
areas of clean chalk.
However in the lab, the experiment separated chlorophyll using a
technique called paper chromatography which was developed in the 1940ís by
the British chemists Archer John Porter Martin and Richard Laurence Millington
Synge, who shared the 1952 Nobel prize in chemistry for their discovery. In
paper chromatography, the stationary phase is a piece of absorbent paper. The
sample is placed as a spot near one end of the paper strip, and this end is dipped
into the liquid that will be the moving phase. The liquid rises by capillary action
and takes the constituents of the sample along with it. A compound with greater
solubility will travel farther than one with less solubility. The substances move at
different rates, depending on their partition between the fixed and moving phases.
To establish the rate of migration, the Rf value for each pigment needs to be
calculated. It is the distance the substance traveled divided by the distance the
developer traveled or Rf = Distance substances (pigments) traveled.
Distance developer traveled
The rate is crucial in finding the connection between polarity and the distance the
pigments traveled. In Part I of the experiment, paper chromatography is used to
separate the polar and nonpolar components of a dye mixture using polar,
nonpolar, and both a polar-nonpolar mixture developers. The polar dyes are
made of ionic compounds which dissolve in water, while the nonpolar dyes do
not. In Part II of the experiment, paper chromatography is used to separate and
observe the plant pigments, chlorophyll a, chlorophyll b, carotenes, and
xanthophylls, that give the leaves their color, calculate their Rf values and the
connection between polar and nonpolar pigments, and their function in
photosynthesis.

Purpose
The purpose of Part I of the experiment, is by using paper chromatography
separate the components of a dye mixture using polar, nonpolar, and both a
polar-nonpolar mixture developers. The purpose of Part II of the experiment, is
using a specially formulated developer, petroleum ether, separate and observe
the plant pigments, chlorophyll a, chlorophyll b, carotenes, and xanthophylls,
which give a leaf its color, calculate their Rf values and the connection between
polar and nonpolar pigments, and their function in photosynthesis.

Hypothesis
In Part I of the experiment, I will prove the connection between polar and
nonpolar dyes and solvents using the separation. In Part II of the experiment, I
will prove that four bands of color will be produced and the pigment that travels
the farthest is the most polar.

Procedure

Equipment Used
In Part I of the experiment the following will be needed:
- 3 chromatography reaction chambers
- 3 chromatography paper strips
- 1 sample loading micropipet
- chromatography dye mixture
- chromatography developer I (deionized water)
- chromatography developer II (isopropyl)
- chromatography developer III (isopropyl/water mixture)
In Part II of the experiment the following will be needed:
- chromatography reaction chamber
- chromatography paper strip
- sample loading micropipet
- chlorophyll extract
- a leaf
- chromatography developer IV (petroleum ether)

Procedure
Part I
First, use a scissors to cut the bottom end of the three chromatography
paper strips ( 2 cm wide X 10.5 cm long) to a tapered end. Second, draw a faint
pencil line to each strip, a few millimeters above the pointed end, and using a
micropipet apply a small drop of the dye mixture on the center of the pencil line to
each strip. Third, pour about 5 mL chromatography developer I (water) into the
reaction chamber. Place one of the chromatography paper strips into the
chamber and adjust the length of the strip so that a small portion of the end tip is
immersed into the developer. DO NOT immerse the pigment spot into the
developer. The top end of the reaction chamber is tapered so that it will hold the
chromatography paper strip at the desired distance. Fourth, ensure that the
paper strip is level and it does not touch the walls of the reaction chamber. Place
the screw cap over the chamber and allow