Title: Thin Layer Chromatography & Pigment Separation in Spinach Leaves

Class Section: CHML-303-Monday

Name: Wail Al-waili
Date Submitted: 04/10/2019

Experimental Procedure/Methods:
Start by Obtaining a TLC plate and a micro capillary. Use a pencil to lightly draw a line
near the bottom and the top of the plate. The lines will be point at which the spinach extract will
be measured. Use the capillary tube to dip one end into the spinach extract. Then empty the tube
in small portions by touching it lightly to the TLC plate at the mark you made. Make a
development chamber from a breaker lined with filter paper and cover. Fill the beaker with about
10-mL of hexane acetone 7.3. Place the spotted plate in the beaker and allow the plate to
develop. The solvent will slowly move up the TLC plate. Remove the plate from the beaker and
using a pencil mark the position of the solvent front. Outline all the observed spots with a pencil.
Finally, measure the distance that each spot has traveled relative to the solvent front.

Results and Observations:

Solvent front (7.1 cm)

Highest non-polar solvent (light-yellow)

Darkest polar solvent (green)

Origin
 Solvent Distance (cm) Rf Value (Species / Pigment/Color
(0 cm – 7.1 cm) Solvent)
A 6.5 0.92 Xanthophyll / yellow
B 3.0 0.42 Xanthophyll / yellow
C 2.3 0.32 Xanthophyll / yellow
D 2.1 0.30 Chlorophyll b/green
E 1.8 0.25 chlorophyll b/green
F 1.7 0.24 Xanthophyll / yellow
G 1.6 0.23 Xanthophyll / yellow
H 1.5 0.21 Xanthophyll / yellow

Discussion:
The separation of mixtures in chemistry can be done using chromatography.
Chromatography is the technique of separating a mixture of two or more different compounds by
their stationary and motile phases. The method depends on the different solubilities. In Thin-
layer Chromatography the stationary phase is the silica, which is in the TLC plate. Silica is
considered the polar substance. Using the capillary tube, the moving phase will be the organic
solvent that will move up the silica-coated-plate.

Basically, we separated the compounds based on polarity. The polar compounds will try to
remain low to the stationary TLC plate while the non-polar compounds will separate and travel
upward with the solvent. As the results shown, the polar compounds raised slowly, and less polar
compounds raised faster. The most non-polar was the very light-yellow pigment, xanthophyll,
which moved up a distance of 6.5 cm.

Conclusion:
In this experiment we performed the separation of pigments in spinach leaves using Thin-
Layer Chromatography (TLC). As a result, the yellow spots on the plate indicate the presence of
xanthophyll and the green spots indicate the presence of chlorophyll b.

References:

Column and Thin-Layer Chromatography of Spinach Extracts,

www.pitt.edu/~ceder/lab5/exp5text.html.