A

Project report

On

TOPIC - Collect the three different plant leaves and separate the
pigments form them by paper chromatography and compare the
result based on Rf values.

Submitted by :

PUSHPRAJ WAGADRE (0901EE231102)

RADHIKA YADAV (0901EE231103)
RAHUL ARORA (0901EE231104)
RAHUL RAWAT (0901EE231105)

Under the guidance of

Dr. Preeti Gupta

Department of Electrical Engineering

Session July Dec. 2024

 CONTENT

Contents
Content.......................................................................................................................................2

1. Introduction.........................................................................................................................3

1.1 Plant Pigments.............................................................................................................................3

1.2 Paper Chromatography............................................................................................................3
2. Materials.............................................................................................................................3

3. Procedure............................................................................................................................4

4. Experimental/data analysis.................................................................................................7

5. Conclusion..........................................................................................................................9

5.1. Chromatogram Report...............................................................................................................9

5.2. Chromatography Conclusion..............................................................................................9
6. Application of paper chromatography..............................................................................10

7. Reference..........................................................................................................................11

8. Declaration by the Candidate............................................................................................12

 1. INTRODUCTION
1.1 Plant Pigments
A compound that absorbs light is called a pigment. Chlorophylls a and b are primary
photosynthetic pigments that absorb light for photosynthesis. The accessory pigments
carotenoids and xanthophyll absorb light and pass it to chlorophyll a. Even though
chlorophyll is the primary pigment, the other pigments are essential to the plant's ability to
produce colour and engage in photosynthesis because they absorb each light differently and
effectively across the electromagnetic spectrum.

1.2 Paper Chromatography

Chromatography, which means "colour writing," is a Greek term that is formed from the
words "chromo" and "graph". Chromatography enables the separation of the constituent parts
of a given mixture, enabling scientists to observe and produce findings and theories.

Paper chromatography is a method for classifying dissolved substances according to how

soluble they are in a given solvent, such as chlorophyll, carotene, and xanthophyll. Paper
chromatography can be used to separate the colours in plant cells. The stationary element in
chromatography paper permits the reaction between the solute and solvent to take place and
produce results.

1.3 Leaf Chromatography

The separation of leaf colours using chromatography is known as leaf chromatography. Leaf
chromatography is an experiment that is conducted to determine the colour of the
photosynthetic pigments. The experiment is conducted to learn about the pigments in the leaf,
and it is mostly done by using paper and thin-layer chromatography
 2. MATERIALS

 Fresh leaves from three different plant species (e.g., spinach, maple, and mint)

 Scissors or mortar and pestle

 Solvent (e.g., a mix of 9:1 petroleum ether to acetone, or rubbing alcohol as a safer option)

 Filter paper or chromatography paper

 Pencil

 Ruler

 Small beakers or glass jars

 Capillary tube or a thin paintbrush

 Ruler
 3. PROCEDURE

1. Prepare Leaf Extracts

 Cut or grind the leaves separately to release pigments.

 Add a small amount of solvent to each sample to extract pigments.

 Filter each extract if there are visible solid particles.

2. Prepare the Chromatography Paper

 Cut three strips of chromatography paper to fit the height of your beakers.

 Draw a light pencil line near the bottom (around 1 cm from the edge) on each strip.
This will be the baseline where you’ll apply the pigment.

3. Apply the Pigment Samples

 Using a capillary tube or paintbrush, carefully apply a small dot of each leaf extract
on the baseline of its respective paper strip.

 Allow each spot to dry and reapply multiple times to concentrate the pigment on the
baseline.

4. Develop the Chromatogram

 Place each strip in a beaker containing a small amount of solvent (below the baseline).

 Allow the solvent to travel up the paper, carrying the pigments with it.

 Remove the strips when the solvent is about 1-2 cm from the top. Mark the solvent
front immediately with a pencil.

 Let the strips dry.

5. Observe and Measure Pigments

 Examine the different bands of color that appear, representing different pigments.

 Measure the distance each pigment traveled from the baseline and the distance of the
solvent front from the baseline.
6. Calculate Rf Values

 Use the formula:

Rf=Distance traveled by pigmentDistance traveled by solvent frontR_f = \frac{\
text{Distance traveled by pigment}}{\text{Distance traveled by solvent front}}Rf
=Distance traveled by solvent frontDistance traveled by pigment

 Calculate Rf values for each pigment band on each chromatogram.

7. Compare Results

 Compare the Rf values of pigments from each plant to identify similarities and
differences.

 Different plants may have unique Rf values for similar pigments (e.g., chlorophyll,
carotenoids) depending on leaf composition.
4. EXPERIMENTAL/DATA ANALYSIS
 5. CONCLUSION

5.1. Chromatogram Report

The final chromatography paper is known as a chromatogram, and it may be photographed to
determine the exact position of each pigment. The pattern of pigment spots on the
chromatography paper at the conclusion of the experiment is called a chromatogram. Along
with the alcohol, the pigments also migrate along the strips of paper.

5.2. Chromatography Conclusion

Carotene is identified as having the lowest molecular weight by its yellow to orange tint near
the top of the paper. In the pigment separation of chlorophyll, chlorophyll may be
distinguished by its blue or dark green hue. When chlorophyll pigments are separated, the
colour yellow-light green identifies chlorophyll B. In the chromatography solvent,
xanthophyll is more soluble since it has gone up the paper. This describes the conclusion of
paper chromatography.

5.3. Final Conclusion

The pigments are light-absorbing molecules and are separated by using paper
chromatography techniques in the lab. The pigments move on the paper based on their
solubility in the solvent. Along with the alcohol, pigments also migrate along the strips of
paper. Some pigments in the leaf travel more quickly than others because of their properties.
 6. APPLICATION OF PAPER CHROMATOGRAPHY

1. Separation of Plant Pigments

 Used in botany and plant sciences to separate and identify pigments in plants, such as
chlorophylls, carotenoids, and xanthophylls. This helps in understanding
photosynthesis and pigment composition in different plants.

2. Food Industry

 Applied to identify and separate food additives, artificial colors, and natural dyes in
processed foods. This ensures food safety and compliance with regulatory standards.

3. Forensic Science

 Employed to analyze inks, dyes, and other compounds in forensic investigations. It

helps in identifying substances in samples from crime scenes, such as drugs or
poisons, based on their unique Rf values.

4. Pharmaceutical Industry

 Used to test the purity of pharmaceutical products and separate and identify active
ingredients in drugs. It helps in quality control and ensures consistency in
pharmaceutical formulations.

5. Environmental Testing

 Applied in analyzing water and soil samples to detect pollutants, pesticides, and
herbicides. Paper chromatography helps identify specific compounds and measure
pollution levels in the environment.

6. Biochemical and Medical Research

 Useful for separating amino acids, nucleotides, and other biomolecules, aiding in
understanding metabolic pathways, enzyme functions, and disease markers. It’s
frequently used in diagnosing metabolic disorders by analyzing biological samples.
 7. REFERENCE

1. Standard Laboratory Manuals and Books on Plant Biochemistry

 "Experiments in Plant Physiology" by Carol Reiss: This lab manual provides

procedures for plant pigment chromatography and Rf value calculations, along with
background information on pigments.

 "Plant Biochemistry" by Hans-Walter Heldt and Birgit Piechulla: Contains details

on plant pigments, including chlorophylls, carotenoids, and their behavior in
chromatography.

2. Journal Articles and Research Papers

 "Separation and Identification of Plant Pigments by Paper Chromatography" in

journals like The Journal of Chemical Education provides detailed methods and
discusses experimental results and analysis of Rf values for plant pigments.

 "Photosynthetic Pigments in Plants: Identification and Estimation through

Chromatographic Methods" in journals like Botanical Studies or Plant Physiology,
which often include discussions on chromatography techniques for pigment
separation.

3. Online Educational Resources

 Science Project Databases like Science Buddies or the American Chemical Society
often have experiments related to chromatography with complete procedures.

 University Websites: Many universities provide online resources, such as the

OpenStax Chemistry for Majors or MIT OpenCourseWare, which include details on
chromatography techniques used in biology labs.
 8. DECLARATION BY THE CANDIDATE

I hereby declare that the work entitled Collect the three different plant leaves and separate
the pigments form them by paper chromatography and compare the result based on Rf
values. is my work, conducted under the supervision of Dr. Preeti Gupta , during the
session Aug-Dec 2024. The report submitted by me is a record of bonafide work carried out
by me.

I further declare that the work reported in this report has not been submitted and will not be
submitted, either in part or in full, for the award of any other degree or diploma in this
institute or any other institute or university.

--------------------------------

PUSHPRAJ WAGADRE (0901EE231102)

RADHIKA YADAV (0901EE231103)

RAHUL ARORA (0901EE231104)

RAHUL RAWAT (0901EE231105)

Date: 14th November 2024

Place: Gwalior
This is to certify that the above statement made by the candidates is correct to the best of my
knowledge and belief.
Guided By:
______________
Dr. Preeti Gupta
Electrical Engineering
MITS, Gwalior