Name: Miriam Harriott

Lab #: 2

Skills Assessed: AI&MM

Date: October 10, 2023

Topic: Photosynthesis

Aim: To separate photosynthetic pigments by chromatography.

Background information:

Green plants employ the process of photosynthesis to turn water and carbon dioxide into food

and oxygen in the presence of light and chlorophyll. Light is absorbed by photosynthetic

pigments during the process of photosynthesis which takes place in the chloroplasts of the

palisade mesophyll and spongy mesophyll tissues found in leaves. The equation for

photosynthesis is 6CO2 + 6H2O + light → C6H12O6 + 6O2.

Chromatography is a technique that can be used to distinguish between and catalog the many

pigments that are present in a plant. A concentrated known extract is applied to a strip of

chromatography paper and then dipped into a suitable solvent in chromatography. The pigments

are separated at various locations on the paper as they are not all equally soluble by the solvent

as it moves up the paper. Chlorophyll A and chlorophyll B are the most prevalent photosynthetic

pigment; they both function by absorbing light wavelengths.

The essential pigment for photosynthesis chlorophyll however plants also contain secondary or

accessory pigments called carotenoids, such as carotene and xanthophyll. Their job is to absorb

light with shorter wavelengths as well as any light that may have evaded chlorophyll’s

absorption. They then transfer this light to chlorophyll. Chlorophyll is shielded from harm by

accessory pigments, which also serves as a type of protection.

Apparatus/Materials: mortar and pestle, beaker, chromatography paper, callaloo leaves,

acetone, ruler, scalpel, capillary tube, pencil, measuring cylinder, stopwatch.

Diagram of setup:

Method:

1) A strip of chromatography paper was cut long enough to reach the bottom of the beaker
and narrow enough that the edges didn’t touch the sides.
2) A ruler was used to measure 30mm from the bottom of the filter paper and a pencil was
used to mark a horizontal line across the paper at 30 mm.
3) A mortar and pestle were used to grind fresh healthy callaloo leaves in 5 ml acetone.
4) A capillary tube was used as a dropper to drop the pigment solution at the center of the
pencil line. The drop of solution was left to dry then a second drop was added and left to
dry. This was repeated 5 more times.
5) Acetone solution was poured into the beaker to a depth of no more than 15mm in height.
6) The chromatography paper was suspended in the beaker. Ensuring that the bottom edge
was dipped into the solvent but the solvent spot was not immersed in the solvent.
7) Results were recorded after 15 minutes.
Results:

DIAGRAM OF THE CHROMATOGRAPHY STRIP SHOWING THE PIGMENTS

Calculations:

Rf =distance travelled by the pigment/distance travelled by the solvent

Green pigment-Rf = 4cm/5.5cm= 0.72

Yellow pigment-Rf = 3.5 cm/5.5cm= 0.64

TABLE SHOWING THE RF VALUES OF A TYPICAL LEAF AND CALLALOO

Name Colour Rf value (typical) Rf value(callaloo)

Carotene Yellow 0.95 0.78
Phaeophytin Yellow gray 0.83 N/a
Xanthophyll Yellow brown 0.71 N/a
Chlorophyll a Blue green 0.65 N/a
Chlorophyll b Green 0.45 0.80
Discussion:

The Rf value of a compound is equal to the distance traveled by the compound divided by the
distance traveled by the solvent front (both measured from the origin). The most polar point is
the one with the lowest Rf value. And the least polar spot has the greatest Rf value. The principal
photosynthetic pigment chlorophyll a, converts light energy into chemical energy that is needed
by the plant. Chlorophyll b sends the energy it creates to chlorophyll a by absorbing light in a
different section of the spectrum than the dominant chlorophyll. The colour of the carotenes and
xanthophylls in the leaves is obscured by chlorophyll since it predominates in green plants.
Chlorophyll, which gives their characteristic green hue, is the most prevalent pigment in leaves.

Phaeophytin, Xanthophyll, and Chlorophyll a pigments were not present in the callaloo as was
shown on the chromatography paper. This is so because the calloloo leaves mostly contains
carotene and the chlorophyll b pigment and as a result it does not absorb a relative amount of the
other pigments wavelength for them to be seen. Carotene absorbs blue-green light reflecting
yellow and it is found around 450 and 475 nm on the absorption spectrum while chlorophyll b
absorbs mainly red-blue light reflecting green light and it is indicated by their peaks around 450-
475 nm and around 650-675 nm respectively. In the comparison of carotene and chlorophyll b as
it relates to a typical leaf the values were 0.95 and 0.45, carotene had an Rf value of 0.78 and
chlorophyll had an Rf value of 0.80.

Based on the Rf values obtained it shows that chlorophyll b in callaloo is more soluble than that
of the typical leaf and carotene is less soluble in callaloo than in the typical leaf. Additionally the
significant difference in values obtained by the Rf values show that the typical leaves contain
more carotene compared to the callaloo leaves which contained chlorophyll b pigments. As
chlorophyll has the largest value, it is clear that it is least polar and moves through the solvent at
a slower rate compared to the two. In other words, carotene the solvents most soluble pigment
would’ve travelled the farthest as it was least soluble in the solvent and chlorophyll b would’ve
moved the least.

Conclusion: Using paper chromatography, the callaloo pigment solution separated into the
photosynthetic pigments chlorophyll b (green) and carotene (yellow).
Limitations:

1) The presence or absence of the other pigments in callaloo, making it unclear what kinds
of pigments to anticipate.
2) The chromatography paper was unable to hold a lot of drops of the pigment without
tearing.

Precautions:

1.) Allow the spots on the chromatography paper to properly dry before placing it into the
beaker with acetone.
2.) The chromatography paper was suspended, not touching the bottom of the beaker.