EXPERIMENT 10

CHROMATOGRAPHY

ALFONSO, GOTERA , LAUSA

PAPER CHROMATOGRAPHY
Paper chromatography is used as an analytical chemistry technique for identifying
and separating colored mixtures like pigments.
Paper chromatography has several components:
Stationary phase
To separate chemicals, we have to have a platform on which to separate them. In our
case, the platform is paper. The paper does not move in the process of
chromatography, therefore, we call it the stationary phase.
Mobile phase
is the solvent that carries the chemicals through the stationary phase. Chemicals dont
move on paper alone, but if we add a solvent, it can draw the chemicals up through
the paper.
Point of origin
the spot where you put your chemicals on the stationary phase.

 Developing
after putting chemicals on the point of origin, the chromatogram can be developed,
that is, the mobile phase can be pulled through the stationary phase.
Retention factor (Rf)
The distance our chemicals move during chromatography is typically less than the
distance the mobile phase moves. We can measure the difference in distance by using
the Rf value. This is the distance a chemical moves on our paper (stationary phase)
during our separation divided by the distance the mobile phase moves
Chromatograph
the finished result from chromatography that shows the separated solute(s) on the
stationary phase after the mobile phase has dried.

SIGNIFICANCE OF PAPER
CHROMATOGRAPHY
Among all the chromatography methods paper chromatography is an
inexpensive and rapid method that provides graphic and clear results.
Used as a qualitative method for identifying the components in a mixture.
The separated spots on the finished and dried chromatogram can be cut out
and re-dissolved to obtain a pure sample of component of the sample
mixtures.
Used in several scientific studies in identification of unknown organic and
inorganic compounds from a mixture.

FACTORS THAT AFFECT RF VALUES:

Solvent concentration
Temperature
The quality of the paper
Solvent purity

OBJECTIVE
To be able to separate compound components flowing across a sorbing
medium
To be able to identify the three colors staining the filter paper
To determine the Rf (rate of flow)value of each color found on the filter
paper.
To know what is chromatography.
To know the procedure of chromatography.
To determine the most effective solvent for chromatography.

MATERIALS
Developer solution
250 ml beaker
Clear cellophane
Rubber band
1 g of leaf sample
10 ml acetone
Mortar and pestle
Filter paper strip

PROCEDURES
Prepare the developer solution
Your instructor will assign the ratio of the solvent system to each group
Pour 30 ml of the developer solution into a 250 ml beaker
Cover the beaker with a clear cellophane with a rubber band tied around it( to
saturate the space above the liquid with vapor)
Weigh 1 g of leaves sample
Cut the leaves into small sizes
Add 10 ml of acetone
Crush the leaves using a clean mortar and pestle

PROCEDURES
Pour the mixture into a vial
Place the vial into a water bath and heat until the volume of the leaves mixture is
reduced to about 1 ml
Obtain a filter paper strip
Draw a pencil line across the paper about 2 cm from the bottom
Along this line, make sample xs about every 4 cm
Spot the chromatography paper at each x mark with the previously prepared leaves
mixture
Place the filter paper in the beaker with the developer
Allow the developer to rise up through the paper

PROCEDURES
When the developer has almost advanced to the top, remove the paper and mark the solvent
point
Allow the paper to dry
There should now be three colors staining the paper
The yellow color is a pigment called carotene
The darker green color is a pigment called chlorophyll a
The lighter green color is a pigment called chlorophyll b
The pinkish/purplish pigment called xanthophyll (but this is often a very difficult substance to
separate.)

Record the color and measure the distances traveled by each colors and the solvent from the
sample line
Determine the Rf value of each color using the equation

 What is the significance of the Rf values?

This value represents the ratio of the distance a pigment traveled on
the chromatogram relative to the distance the solvent front moved.
Scientists use the Rf value of a sample to identify the molecule.

An Rf value that is close to 1 indicates that the pigment is very soluble in

the solvent.
An Rf value that is very small indicates that the pigment is not that soluble
in the solvent.

PRE LAB ASSIGNMENT

Write the structural formula for carotene, chlorophyll A, chlorophyll B
Carotene ( C40H56 )

-Carotene is a strongly colored red-orange pigment abundant in

plants and fruits. It is an organic compound and chemically is
classified as a hydrocarbon and specifically as a terpenoid, reflecting
its derivation from isoprene units.

PRE LAB ASSIGNMENT

Write the structural formula for carotene, chlorophyll A, chlorophyll B
Chlorophyll A (C55H72O5N4Mg )

Chlorophyll a absorbs most energy from wavelengths of violet-blue and

orange-red light. It also reflects green/yellow light, and as such contributes to
the observed green color of most plants. This photosynthetic pigment is
essential
for
photosynthesis
in
eukaryotes,
cyanobacteria
and
prochlorophytes because of its role as primary electron donor in the electron

PRE LAB ASSIGNMENT

Write the structural formula for carotene, chlorophyll A, chlorophyll B
Chlorophyll B (C55H70MgN4O6)

Chlorophyll b helps in photosynthesis by absorbing light energy. It is more

soluble than chlorophyll a in polar solvents because of its carbonyl group. Its
color is yellow, and it primarily absorbs blue light.

WHAT IS THE DIFFERENCE BETWEEN THE

STRUCTURAL FORMULA OF CHLOROPHYLL
A AND CHLOROPHYLL B?
Chlorophyll A

Chlorophyll B

Principal photosynthetic pigment

Present in all phototrophs other than
bacteria
Blue green in pure state
Empirical formula C55H77O5N4Mg
The 3rd carbon of the side group is methyl
group
Molecular weight is 873

Accessory photosynthetic pigment

Present in all phototrophs other than
diatoms, cyanobacteria, red and
brown algae
Olive green in pure state
Empirical formula C55H70O6N4Mg
Side group at the 3rd carbon is an
aldehyde group
Molecular weight is 907

PRE LAB ASSIGNMENT

What vitamin is related to carotene?
Biologically, beta-carotene is most important as the
precursor of vitamin A in the human diet. It also has antioxidant properties and may help in preventing cancer and
other diseases.
Vitamin A has several functions in the body. The most well
known is its role in vision - hence carrots "make you able to
see in the dark". The retinol is oxidized to its aldehyde,
retinal, which complexes with a molecule in the eye called
opsin. When a photon of light hits the complex, the retinal
changes from the 11-cis form to the all-trans form, initiating
a chain of events which results in the transmission of an
impulse up the optic nerve

DEVELOPING SOLVENTS USED

DATA AND RESULTS

Results:
Solvent 1: Carotenoids- Most soluble; Chlorophyll a- Least soluble
Solvent 2: Chlorophyll a- More soluble; Chlorophyll b- least soluble

Name of Sample: Lagundi

Developing solvent: Solvent 1: 9 hexane, 1 EtAc || Solvent 2: 7hexane, 3EtOH
COLOR

DISTANCE TRAVELLED BY
SOLUTE
SOLVENT
(COLOR)

Rf

Carotenoid (yellow)

6.5cm || ---

8.5cm || 7.4cm

0.764 || ---

Chlorophyll a (dark
green)

3.0cm || 4.3cm

8.5cm || 7.4cm

0.352 || 0.581

Chlorophyll b (light
green)

5.2cm || 3.6cm

8.5cm || 7.4cm

0.612 || 0.424

Xanthophyll
(pink/purple)

---

---

---

POST LAB QUESTIONS

What would be the consequence of having so much solvent in the
developing chamber that it would cover the application spot?
It would dissolve the spot, so youd have to start over. The chamber should
contain just enough solvent to cover the bottom

POST LAB QUESTIONS

What effect would it have on the chromatogram with a broad sample spot?
The chromatogram could be messy, overlapping of spots can occur, making it
hard to determine the distance your sample traveled

POST LAB QUESTIONS

How is thin layer chromatography technique of separation similar to a paper
chromatographic separation?
In both types of chromatography, samples are placed near the bottom of the
stationary phase which is then set into a beaker containing a small amount of
the mobile phase, which travels up the stationary phase via capillary action. The
samples travel different distances up the stationary phase depending on their
polarity. This is also useful for separating the components of one sample.

CONCLUSION
Plants differ in the photosynthetic pigments present in them. These pigments have
varying properties which were investigated in this experiment.
Paper chromatography proved to be an accurate method of separating and
observing the various colors of plant pigments. The pigments dissolved in the
solvent and migrated upward. The colors were observed and their migration
distances measured & recorded. The R f value of each pigment was determined by
dividing its migration by the migration of the solvent. It was determined that 3
pigments were present in the original spot --- carotene, chlorophyll a, and
chlorophyll b. Carotene was the most soluble, while chlorophyll b was the least
soluble.
These results were due to the polarity of the pigment chlorophyll, which is an
organic compound, in the same types of compounds such as acetone.