Indian Institute of Technology Jammu

Department of Chemical Engineering

SBP001U3M
BIOLOGY FOR ENGINEERS
III YEAR, FIFTH SEMESTER, 2021

EXPERIMENT NO- Thin

Layer chromatographic
separation of Spinach and
White Radish leaves
extract
MEMBERS:PIYUSH VERMA (2019UCS0106)

Experiment carried on: December, 2021

Report submitted on : December, 2021
EXECUTIVE SUMMARY (05) ______
INTRODUCTION/OBJECTIVES/SCOPE/PROCEDURE (25) ______

RESULTS & DISCUSSION (40) ______

a) Data analysis and interpretation of information

b) Presentation of relevant information (including results
on graphical, tabular or equation forms)
CONCLUSIONS (05) ______
REFERENCES (05) ______
APPENDIX
a) Original data, sample calculations, BioCOSHH (05) ______
GENERAL COMPLETENESS
a) Conciseness and neatness (05) ______
VIVA-VOCE (10) ______

TOTAL (100) _____

FEEDBACK SHEET
EXECUTIVE SUMMARY:
Plants that are photosynthetic transform the sun's light energy into chemical food energy.
Pigments are utilised to capture light energy during photosynthesis. Pigments are chemical
substances that reflect just certain visible light wavelengths. Chlorophyll a (dark green),
chlorophyll b (yellowish-green), xanthophylls (yellow), and carotenoids are the principal
pigments found in plant leaves (orange). With the use of thin layer chromatography, we will aim
to extract and separate the various plant pigments present in fresh spinach leaves and white
radish leaves in this experiment (TLC). We'll mill the spinach and radish leaves separately until
they're a light green hue. After that, remove 2-3 ml from the ground combination of each
samples.

After that, take a TLC plate with a dimension of 3.5 cm* 9.0cm and draw a 1cm line across the
bottom. Transfer the extract as usual, then elute the plates in a closed chamber with a mobile
phase (a 60 percent petroleum mixture) wait until the separation is complete (ether, 26% acetone,
10% ethyl acetate, 4% methanol) We were able to see different colours in the form of after the TLC
plate had dried. Various coloured specks at various levels on the plate.

INTRODUCTION:
Pigments are defined as a group of substances with a bright hue that are used to colour other
materials. These pigments are also referred to as Biological Pigments or Biochromes, which
refers to the actual pigments. In this project, we'll use thin layer chromatography to extract and
separate various pigments from spinach and radish leaves using the procedure outlined below.

OBJECTIVE:
To extract and separate the chlorophyll a and b from spinach and radish leaves by thin layer
chromatography.

PROCEDURE:
•Take a handful of green spinach leaves that have just been harvested.

• Tear the spinach leaves into small chunks and drop them into the mortar.

• Using a mortar and pestle, grind the spinach leaves for 5-10 minutes.

• Fill a tiny test tube halfway with acetone and the green solid grinded mixture.

• Now stir the heterogeneous mixture to ensure that the acetone and solid are completely mixed.

• Take a piece of filter paper with a narrow notch on one end.

• Using a pencil and a scale, draw a horizontal line about 2-3 cm away from the notch's tip.
• Using a capillary tube, place a drop of the pigment extract in the middle of the line.

•Allow the drop to dry before placing another 2 to 3 drips on the TLC plate.

• Fill the chromatographic chamber with a mobile phase made up of 60 percent petroleum ether,
26 percent acetone, 10% ethyl acetate, and 4% methanol.

• Keep the loading spot about a centimetre above the solvent level.

• Allow some time for the chromatographic chamber to be undisturbed.

• We can see how the solvent spreads the different colours in the mixture to different distances as
it goes through the silica plate.

• When the solvent has risen about 3/4 of the way up the strip, carefully remove it and let it dry.

The radish leaves are prepared in the same way.

RESULT & OBSERVATION:

The xanthophylls are indicated by the yellowish patch near the top. Chlorophyll an is represented
by the dark green speck second from the top. Chlorophyll b is seen in the lowermost yellowish
green patch.
The equation can be used to calculate the R f value of each pigment spot.

R_f=(Distance travelled by the compound)/(Distance travelled by the solvent)

The distance of each pigment band was measured from the loading spot and also the distance
travelled by the solvent.

PIGMENTS Chlorophyll a Chlorophyll b b xanthophylls

SPINACH 0.68 0.59 0.93

RADISH 0.58 0.48 0.94

CONCLUSION:
Three separate spot bands can be seen on the dried TLC strip. Color can be used to distinguish
different pigments, such as "chlorophyll a" (dark green spot) in the centre, "chlorophyll b"
(yellowishgreen spot) at the bottom, and "xanthophylls" (yellow spot) at the top.

RECOMMENDATION:
• Spinach leaves should be fresh and green.

• The loading spot should be 2-3 cm away from the tip of the notch.

• While placing the TLC plates in the chromatography chamber, the loading spot
should remain about 1 cm above the solvent level.

REFERENCES:

https://en.wikipedia.org/wiki/Thin-layer_chromatography

https://www.sigmaaldrich.com/IN/en/applications/analytical-chemistry/thin-
layer-chromatography
https://www.sciencedirect.com/topics/materials-science/thin-layer-
chromatography