NAME:Vasudev Chanka

DATE: 6/Jan/2022

Module 2: Analytical Methods and Separation Techniques

TITLE: Analysis of Spinach Leaves using Thin Layer Chromatography.

AIM: To identify the pigments present in spinach leaf extract using thin layer chromatography.

THEORY: Thin-layer chromatography is a chromatography technique used to separate non-volatile

mixtures. The mobile phase is a suitable liquid solvent or mixture of solvents.The mobile phase flows
through the stationary phase and carries the components of the mixture with it. Different
components travel at different rates.Thin layer chromatography (TLC) is an affinity-based method
used to separate compounds in a mixture. TLC is a highly versatile separation method that is
widely used for both qualitative and quantitative sample analysis.

MATERIALS: TLC developing solvent (hexane acetone), spinach juice, pentane

APPARATUS: 50 ml beaker, 2 graduated cylinder, pasteur pipet, 2 10cm screwed cap test
tubes, test tube rack,hot plate, TLC plate, pencil, ruler, forceps, developing chamber,
microcapillary tubes, TLC developing solvent, spinach juice, pentane.

PROCEDURE:

1) 3 mL of spinach juice was added to the screwed cap test tube

2) Six ml of pentane was added to the test tube
3) The test tube was capped and was shaken vigorously for one minute
4) Sample test tube was placed in position one in the centrifuge
5) A second screwed cap test tube was filled with water equal to the amount of sample and it was
placed directly opposite the sample to keep the load balance
6) The lid was closed and timed for 2 mins
7) The test tubes were removed from the centrifuge and the separation was observed in the sample
test tube
8) A pasteur pipette was used to transfer the dark green tab layer to a clean 50 mL beaker
9) The pentane was evaporated by heating the beaker on a hot plate at a low setting about 95°C for
three minutes
10) When 1 to 2 mL of liquid remained, the beaker was removed from the hot plate immediately to
prevent degrading of the Spinach pigment
11) 5 mL of the TLC developing solvent and a 7 to 3 mixture of hexane acetone was added in the
developing chamber
12) The developing chamber was stopped to allow it to become saturated with solvent chambers. It
was Ensured that the depth of the solvent is no more than 0.5 cm
13) five minutes was taken to allow the chamber to equilibrate before the first tlc plate was
developed
14) A light pencil line was drawn 1 cm from the bottom of the tlc plate. This was the Start line
15) Another light pencil line was drawn 5cm above the first line. This was the finish line
16) A small tick line was drawn in the centre of the start
17) A microcapillary tube was used to load the extract onto the tlc plate.
 18) The tip of the draft at the end of a Microcapillary tube was allowed to just touch the plate at the
small checkmark in the centre of the start line.
19) The solvent was allowed to evaporate between drops
20) It was ensured the drops were consistent
21) The drops were allowed to dry
22) Forceps was used to place the tlc plate into the developing chamber cap immediately to avoid loss
of the solvent saturated atmosphere
23) How quickly the solvent began to migrate up the olate was observed
24) As soon as the solvent had reached the top line on the Tlc plate. Forceps was used to remove the
plate. The plate was then placed on the bench to dry
25) As the plate had dried a change was observed in its appearance. The spot that had pigment or
colour material was observed. Spots without pigment was known to not be visible
26) The dried tlc plate was placed under the uv light
27) An outline of each spot on the plate was gently drawn.

RESULTS: DISTANCE TRAVELLED AND Rf VALUE OF EACH SPOT

COLOUR OF SPOT DISTANCE TRAVELLED Rf value

(CM)

yellow 0.90 0.18

green 1.50 0.30

blue 1.80 0.36

grey 2.30 0.46

orange 4.30 0.86

DRAWING OF THE CHROMATOGRAM

CALCULATIONS:

Retention factor = distance travelled by spot/ distance travelled by solvent

Yellow spot = 0.9/5 = 0.18

Green spot = 1.5/5 = 0.3

Blue spot = 1.8/5 = 0.36

Grey spot = 2.3/5 = 0.46

Orange spot = 4.3/5 = 0.86

DISCUSSION:

pentane may be added to redissolve the green residue.Adding a drop or two of pentane after
evaporation will ensure better loading of the TLC plate. The solvent was left to equilibrate in the
chamber, because You want your chamber air thoroughly filled with solvent vapour in TLC
because this keeps the stationary phase from drying out before the process is finished. If the
stationary phase dries out prematurely, the components in the sample won't separate properly
and your results will be incorrect.If you have an over-large spot, this could cause overlapping of
other component spots with similar Rf values on your TLC plate. If overlapping occurs, it would
prove difficult to resolve the different components. A small, concentrated spot is always
preferred. In thin layer or paper chromatography the origin should be above the solvent
because it could otherwise wash the sample spot into the solvent trough.

the chosen solvent system was considered to be polar

Beta carotene - orange spot

Pheophytin - grey

Chlorophyll A - blue

Chlorophyll B - green

Xanthophyll - yellow

CONCLUSION:

The pigments present in an unknown sample using thin layer chromatography were Beta carotene,
Pheophytin, Chlorophyll A, Chlorophyll B, and Xanthophyll

REFERENCES: https://sites.google.com/ncsu.edu/ncstatevrorganicchemistrylabs/home

https://player.wondavr.com/p/a2455203-6d18-4361-b144-148de452d089