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LAB 6 :


The technique helps in analysing, identifying, purifying and quantifying
unknown separable mixtures. The mobile phase is either a liquid or gas which
moves the solvent through the stationary phase during the process. The
stationary phase is a liquid or solid component that is fixed in a place for the
procedure. Paper chromatography works majorly on capillary attractions. The
capillary attraction which depends on adhesive and cohesive forces allows the
mobile phase to move up the stationary phase due to created surface tension
interaction from the forces. The major types are the paper chromatography, thin
layer, gas chromatography, column chromatography, high performance liquid
chromatography, paper chromatography and thin layer chromatography. There
are several applications of paper chromatography and other main types of
chromatography techniques.

1. To prepare an extract of plant tissue
2. To apply the technique of paper chromatography as a method of separating
individual plant pigments contained in plant tissue extracts containing pigment
3. To identify the presence of different pigments in chlorophyll's.
4. To identify the Rf .

1. Test tube
2. Cork stopper
3. Pin
4. Chromatography paper
5. Solvent
6. Pencil
7. Ruler

1. Used pin microbiurette /pin head as the dropper, dropped the chloroplast
extract on prepared chromatography paper.
2. The extract was dropped at about 1.0cm from the pointing end of the paper /
The drop was dried with a hair dryer and the process were repeated for 3-4
times until one small dot of thick pigment available.
3. The paper strip was attached at the cork stopper using a pin . The strip was
placed vertically and straight into the test tube which contained solvent.
4. Let the solvent moves and the paper was removed before the solvent front
reaches the top of your chromatography paper .
5. Marked with pencil the last range of the solvent.


Red bayam Green bayam

Rf = distance travelled by compound/
distance travelled by solvent

Red bayam Green bayam

Rf = 8.5/9.8 Rf = 7/9.2
= 0.87cm = 0.76cm

1. Why the developing solvent mixture is prepared fresh before use ?
The developing liquid phase comprises of a pure solvent but more often it is a
mixture of two or more solvents in specified proportions. In case solvents are
mixed and stored for long periods there could be loss of volatile component
which will alter the mixing proportions.

2. Why is it important to keep the dye spot (leaf extract) above the solvent level?
It is important to keep the dye spots above the solvent level because if the dye
spots of submerged in the solvent, then the spots would dissolve into the solvent
preventing them from separating out and no measurements or observations could
be made.

3. Why is it necessary to cover the test tube during the paper development ?
During the chromatogram development chamber is covered. This is essential as
the environment inside the chamber should remain saturated with the solvent
vapour. Development times can vary from about an hour to several hours and a
saturated environment prevents losses due to evaporation

4. Why is it important to stop the chromatogram before the solvent front reaches
the top of your chromatography paper ?
It is important to stop it because you cannot determine the Rf value unless you
can measure the distance from the start to the front of solvent, you have to be able
to see where it stops even if you let it go as high as you want.
5. Why is it important to mark the solvent level on the chromatography paper
when you remove it from the test tube ?
It is important to mark the solvent level on the chromatography paper when you
remove it form the test tube because so the point at which the solvent stopped
could be noted in case the solvent kept advancing when removed

6. Which of the pigments migrated the farthest and why the separation of
pigments occur as it did ? How does paper chromatography work ?
Those pigment molecules that have highest solubility in the liquids solvent used
will be carried along through the paper the fastest . Separation of pigments occur
because it is based on their level of hydrophobic and hydrophilic properties .
Simple chromatography is carried out on paper. A spot of the mixture is placed
near the bottom of a piece of chromatography paper and the paper is then placed
upright in a suitable solvent . As the solvent soaks up the paper, it carries the
mixtures with it. Different components of the mixture will move at different rates.
This separates the mixture out.

7. Explain what would happen to your chromatogram if you let it run too long ?
If you allowed your chromatogram to develop too long the solutes and solvent
would travel too far and possibly run of the plate. Therefore we would not be able
to calculate the Rf values without a measured solvent front

After completing this lab, each pigment present was separated and identified
based on its solubility to the solvent and the R F value of each pigment was
determined based on their distance travelled. Each pigment moved up the
chromatography paper based their solubility and was measured with a ruler
from the line of origin to the point where the pigment stopped traveling up. Due
to procedural error, we submerged the chromatography paper too far in the
solution instead of letting it sit close to the top of the solvent and letting the
solvent move up the paper. The Rf values were found to be 0.87 for red bayam
and 0.76 for green bayam .