S.P KIRAN KUMARI* et al.

ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

SEPARATION AND OBSERVATION OF PLANT PIGMENTS IN

FERTILIZERS EFFECTED MEDICINAL PLANTS USING PAPER
CHROMATOGRAPHY

S.P Kiran kumari 1, Y. Satya vani 2, V Sridevi 3, M.V.V. Chandana Lakshmi 4

1
M.Tech Biotechnology, Dept of Chemical Engineering, Andhra University, A.P, India, kirank2306@gmail.com
2
M.Tech, Dept of Chemical Engineering, Andhra University, A.P, India, satya.yadla@gmail.com
3
Associate professor, Dept of Chemical Engineering, Andhra University, A.P, India, vellurusridevi@yahoo.co.in
4
Associate professor, Dept of chemical Engineering, Andhra University, A.P, India,mahantilakshmi@yahoo.com

Abstract
Plants are one of five big groups of living things. The whole plant biodiversity is the largest source of herbal medicine and among
these India have 15 agro climatic zones and 17000- 18000 species of flowering plants of which 6000-7000 are estimated to have
medicinal usage. The Indian medicinal plants and their products also account of exports in the range of Rs. 10 billion. It raises a need
of studies in medicinal plants as these are involved in maintaining traditional therapies and also they lead to economical support to
the country. In this research paper three different varieties of medicinal plants are subjugated to fertilizers treatment for 60 days and
later on the plant pigments were separated using paper chromatography and observed under UV-Transilluminator. The Rf values
were noted down and compared with their respective control plant values .The presence or absence of the pigments when compared to
the control plant significantly indicates the effect of fertilizers on the medicinal plant pigments.

Index Terms: Medicinal plants, Fertilizers, Plant pigments, Paper Chromatography, UV-Transilluminator
-------------------------------------------------------------------------*** ---------------------------------------------------------------------

1. INTRODUCTION
Medicinal plants are an accessible, affordable and culturally
appropriate source of primary health care for more than 80%
of Asia's population (WHO) [1]. Marginalized, rural and
indigenous people, who cannot afford or access formal health
care systems, are especially dependent on these culturally
familiar, technically simple, financially affordable and
generally effective traditional medicines [2]. As such, there is
widespread interest in promoting traditional health systems to
meet primary health care needs [3]. This is especially true in
South Asia, as prices of modern medicines spiral and
governments find it increasingly difficult to meet the cost of
pharmaceutical-based health care. Throughout the region,
there is strong and sustained public support for the protection
and promotion of the cultural and spiritual values of traditional
medicines [4]. Many of the modern medicines are produced
indirectly from medicinal plants. Plants are directly used as
medicines by a majority of cultures around the world.
Cultivation and preservation of medicinal plants protect
biological diversity [3].
There cultivation plays a vital role in its high scale production
of medicinal plants. Fertilizers is any organic or inorganic
material of natural or synthetic origin (other than liming
materials) that is added to a soil to supply one or more plant
nutrients essential to the growth of plants [5]. The use of
synthetic nitrogen fertilizers has increased steadily in the last
50 years, rising almost 20-fold to the current rate of 100
million tonnes of nitrogen per year [6].The use of phosphate
fertilizers has also increased from 9 million tonnes per year in
1960 to 40 million tonnes per year in 2000.Organic fertilizers
include naturally occurring organic materials like manure,
worm castings, compost, seaweed, guano or naturally
occurring mineral deposits. Organic fertilizers have been
known to improve biodiversity and long-term productivity of
soil [7] [8], and may prove a large depository for excess
carbon dioxide [9] [10] [11].There are various benefits of both
organic and inorganic fertilizers, and for analyzing that we are
going to conduct this experimental research work to check out
the effect of fertilizers on the plant pigments.

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 317
S.P KIRAN KUMARI* et al. ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

1.1 Medicinal plants subjugated for the research
work
Three different varieties of medicinal plants were collected
from the nursery and subjected for treatment with fertilizers.
Three plants of each variety were collected, among those three
one plant acts as a control and other two plants are treated
with organic and inorganic fertilizers for 60 days after sown.
Later on, these plant samples were collected for the plant
pigment separation by using paper chromatography technique.
The Plant of our choice was Azadirachta indica, Cassia
fistula, Catharanthus roseus as they possess lot of medicinal
value.
1.2 Selected Medicinal Plant importance
Azadirachta indica is a fast growing, long-lived tree with
unpleasant smelling wood that grows to about 12 meters. It
has evergreen pinnate leaves and small fragrant yellow-white
flowers, followed by green-yellow berries [12]. It is a bitter
tonic herb that reduces inflammations and clears toxins, while
promoting healing and improving all body functions. Apart
from this it destroys a wide range of parasitic organisms and is
also insecticidal and spermicidal.
Cassia fistula is native of India, commonly known as
Amaltaas, is one of the most beautiful of all tropical trees
when it sheds its leaves and bursts into a mass of long, grape-
bunches like yellow gold flowers. A tropical ornamental tree
with a trunk consisting of hard reddish wood, growing up to
40 feet tall [13].

Catharanthus roseus is a fleshy perennial growing plant

which grows to 32 inches (80 cm) high. It has glossy, dark
green, oval leaves (1-2 inches long) and flowers all summer
long. The blooms of the natural wild plants are a pale pink
with a purple "eye" in their centers
[15] [16]. In the wild, it is an endangered plant and the main
cause of decline is habitat destruction by slash and burn
agriculture [17]. It is also however widely cultivated and is
naturalized in subtropical and tropical areas of the world [18].

The Importance of these respective plants is as many as we

can list out, but some of them are listed out and tabulated in
Table-1.

Table-1: Details and application of the medicinal plants

which are involved in the research work

Medicinal Family Involved in various

plants treatments
Cassia fistula Fabaceae It is also called as disease
killer" and useful curing skin
diseases, burning sensations ,
syphilis, cardiac disorders,
intermittent fever and general
debility
Meliaceae Involves in curing fever,
nausea, vomiting, treat
Azadirachta malaria, tuberculosis,
indica arthritis, intestinal worms, as
well as to treat skin disease.
Figure-1: A- Cassia fistula, B- Azadirachta indica,C-
Catharanthus roseus

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 318
S.P KIRAN KUMARI* et al.
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY
Apocynaceae It arrest bleeding, gargling
may help sore throats and
Catharanthus chest ailments, laryngitis
roseus and the plant contains
alkaloids Vinblastine and
vincristine, which are used
in the treatment of tumor,
leukemia, hypertension and
has a sedative property.
2. TECHNIQUES INVOLVED IN THE
SEPARATION OF PLANT PIGMENTS
2.1 Paper chromatography
Chromatography is the collection term for a set of laboratory
techniques for the separation of mixtures. The mixture is
dissolved in a fluid called the “mobile phase”, which carries it
through a structure holding another material called the
“stationary phase”. The various constituents of the mixture
travel at different speeds, causing them to separate. The
separation is based on differential partitioning between the
mobile and stationary phases. Subtle differences in a
compound’s partition coefficient result in differential retention
on the stationary phase and thus changing the separation.
Paper chromatography is a technique that involves placing a
small dot or line of sample solution onto a strip of
chromatography paper. The paper is placed in a jar containing
a shallow layer of solvent and sealed. As the solvent rises
through the paper, it meets the sample mixture which starts to
travel up the paper with the solvent. This Paper is made of
cellulose, a polar substance, and the compounds within the
mixture travel farther if they are non–polar. More polar
substance, bond with the cellulose paper more quickly and
therefore do not travel as far [19].
IJESAT | Mar-Apr 2012
Available online @ http://www.ijesat.org
ISSN: 2250–3676
Volume-2, Issue-2, 317 – 326
Fig-2: Paper chromatography for separating the colored
samples
Photosynthesis is the main metabolism for the survival of
plants. Photosynthesis is the process which transforms light
energy from the sun into chemical energy for the plants. This
mechanism is done by the presence of plant pigments which
are the macro molecules produced by the plants. These
pigments absorb specified wavelengths of visible light to
provide the energy required for photosynthesis. Chlorophyll is
necessary for photosynthesis, but accessory pigments collect
and transfer energy to chlorophyll. Although pigments absorb
light, the wavelengths of light which are not absorbed by the
plant pigments are reflected back. Plant contain different
pigments and some of pigments observed include are
Chlorophylls (green), Carotenoids ( yellow , orange-red) ,
Anthocyanins (red to blue, depending on pH), Betalains (red
or yellow).
In Paper chromatography, paper is marked with the plant
extract and is placed in a developing chamber with a specified
solvent. The solvent carries the dissolved pigments as it moves
up the paper. This shows the variation of the pigments from
one fertilizer effected plant to another and finally compared
with the control plant pigments. The distance of the pigment
travels is unique for the pigment in set conditions and is used
to identify the pigment. The ratio is the retention factor Rf
value.
The distance travelled by the pigment (cm)
Rf= _____________________________________
The distance travelled by the solvent (cm)
2.2 UV-Trans illuminator
Ultraviolet (UV) light is electromagnetic radiation with a
wavelength shorter that of visible light but longer than X rays,
in the range of 10nm to 400 nm, and energies from 3eV to 124
eV. It is named because the spectra consist of electromagnetic
waves with frequencies higher than those that human identify
as the color violet. These frequencies are visible to humans,
but visible to a number of insects and birds. They are also
indirectly visible by causing florescent materials to glow with
visible light. The electromagnetic spectrum of ultraviolet light
can be subdivided in a number of ways. The draft ISO
standard on determining solar irradiances (ISO-DIS- 21348)
describes the following ranges which are tabulated in the
Table-2[20] [21].
319
S.P KIRAN KUMARI* et al. ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

Table-2: Electromagnetic spectrum of Ultra violet light 3. MATERIALS AND METHODS

Name Abbreviation Wavelength Energy per 3.1 Plant cultivation
range photon
Cassia fistula, Azadirachta indica, Catharanthus roseus, each
In
variety three plants were collected from nursery and plant in
nanometers
the allotted land. Daily these were watered and two plants
Before Visible light Over 400 Under 3.10 from each were treated with inorganic and organic fertilizers
UV nm eV at a dosage of 1gm/ml. These plants were subjugated for this
spectrum treatment for 60 days after grown. Mean while, the control
plant of each variety was just grown without any chemical
Ultraviolet UVA 400-315 nm 3.10-3.94 eV treatment. Later on the plants were ready for the experimental
A ,Long work.
wave, or
Black
light
Near NUV 400-300 nm 3.10-4.13 eV
Ultraviolet MUV 300-200 nm 3.94-4.43 eV
B or
medium
wave
Middle MUV 300-200 nm 4.13-6.20 eV

Ultraviolet UVC 280-100nm 4.43-12.4 eV

C, short
wave, or
germicidal

UV transilluminators consist of a UV light source and

associated electronics housed in a box with appropriate optical
filters. For safety purpose it has a light transmitting cover with
UV filter that transmits visible light to allow specimen to be
viewed. All UV trans-illuminator are major sources of UV
radiations which can cause damage to skin and eyes of an
individual. Due to its major disadvantage of causing damage
to humans, in future it will be replaced by blue rays and dyes
[22].

Fig-3: UV-Transilluminator

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 320
S.P KIRAN KUMARI* et al.
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY
Fig-4: All the three varieties of medicinal plants, I- Cassia
fistula, II- Azadirachta indica, III- Catharanthus roseus
A- Control plant; B- Inorganic fertilizer treated
plant; C-Organic fertilizer treated plant
3.2 Plant sample preparation
The plant samples from each plant are collected and cleaned
twice by using water as shown in Fig-5.In order to extract the
pigments from the thylakoid membranes of the chloroplast,
fresh, grounded leaves may be soaked in acetone or
concentrated alcohol. The chloroplast pigment was extracted
in 95 % ethanol for several minutes and then filtered using
gravity filtration.
Fig-5: Cleaned plant leaves
IJESAT | Mar-Apr 2012
Available online @ http://www.ijesat.org
ISSN: 2250–3676
Volume-2, Issue-2, 317 – 326
3.3 Developing Paper chromatography
 Cut a piece if whatman No.1 filter paper or
chromatography paper to the dimensions of 18cm
long whereas one of the edge must be blunt and other
end should be pointed.
 With a pencil lightly make a line of 1.5-2cm from
pointed edge of the paper.
 Take the extracted plant pigment was painted onto
the strips of chromatography paper with the help of
V- shaped tips or glass tubes or by using small paint
brush.
 Allow the line to dry, and then repeat the process
until a dark green line of pigment is evident to see.
 Next, Chromatography solvent is used to separate the
mixture of pigments painted on the paper.
 Chromatography solvent is prepared by adding nine
parts of petroleum ether to one part of acetone.
 This chromatography solvent is highly volatile and
flammable.
 The V-Shaped tip of the paper is placed in the
chromatography solvent and acts as a wick to draw
the solvent up the paper, separating pigments
according to their relative solubility and molecular
weights.
 The paper is allowed to remain in the solvent until
the uppermost pigment band nears the top of the
paper.
Fig-6: Developing chromatogram
3.4 Visualizing using UV-Transilluminator
After running the chromatographic sample, those strips were
visualized under the effect of UV- Transilluminator. Whereas
it has three parameters to study, we can visualize the strips
321
S.P KIRAN KUMARI* et al. ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

under day light, long wave length (UVA) and short wave
length (UVC) and the differences were recorded.
Fig-8: Azadirachta indica Paper Chromatography
development , C-Control, O-Organic effected plant , I-
4. RESULTS AND DISCUSSION Inorganic effected plant
4.1 Paper chromatography development results for
research subjugated plant samples
All the plant samples were run down for paper
chromatography, Cassia fistula, Azadirachta indica,
Catharanthus roseus, showed various differences in height
and pigments visualizing too. All the chromatography strips of
these three plant samples are shown below in Fig-7, 8, 9.

Fig-9: Catharanthus roseus Paper Chromatography

development, C-Control, O-Organic effected plant, I-
Inorganic effected plant

Fig-7: Cassia fistula Paper Chromatography development,

C-Control, O-Organic effected plant , I-Inorganic effected 4.2 Visualization of Paper chromatography strips
plant
under UV- Transilluminator
Now the paper chromatography developed strips of each
variety of medicinal plant are visualized under UV-
Transilluminator under short and long wavelengths and some
sort of pigments were visualized only under long UV
wavelengths. Fig-10, 11, 12 shows the bands of Cassia fistula
control plant, organic fertilizer effected plant, inorganic
fertilizer effected plant.Fig-13, 14, 15 shows the colored bands
of Azadirachta indica under the effect of UV short and long
wave lengths.Fig-16, 17, 18 shows the colored bands of
Catharanthus roseus under the effect of Short and long
wavelengths of UV.

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 322
S.P KIRAN KUMARI* et al. ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

Fig-10: Visualization of Cassia fistula control plant Fig-13: Visualization of Azadirachta indica control plant
pigments under both short and long wave lengths of UV pigments plant pigments under both short and long wave
lengths of UV

Fig-14: Visualization of Azadirachta indica organic

Fig-11: Visualization of Cassia fistula organic fertilizer fertilizer effected plant pigments under both short and
effected plant pigments under both short and long wave long wave lengths of UV
lengths of UV

Fig-15: Visualization of Azadirachta indica inorganic

Fig-12: Visualization of Cassia fistula inorganic fertilizer fertilizer effected plant pigments under both short and
effected plant pigments under both short and long wave long wave lengths of UV
lengths of UV

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 323
S.P KIRAN KUMARI* et al. ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

Fig-18: Visualization of Catharanthus roseus inorganic

fertilizer effected plant pigments under both short and
long wave lengths of UV

4.3 Retention factor value of the developed plant

Fig-16: Visualization of Catharanthus roseus control plant
pigments under both short and long wave lengths of UV
pigments
Based on the bands formed on the chromatography paper, the
retention factor Rf value are calculated for each type of
pigment. This is done by dividing the distance travelled by the
pigment and distance travelled by the solvent. The values are
tabulated in the Table-3.
Table-3: Rf values of the three different medicinal plants
which are grown under three different conditions are
tabulated.

Medicinal Rf value of Rf value of Rf value of

plants Control Organic Inorganic
plant fertilizer fertilizer
effect effect
Cassia fistula 0.7- 0.74- 0.52-
Xanthophyll Xanthophyll Xanthophyll
pigment pigment pigment
0.31- 0..64- 0.70-
Chlorophyll Chlorophyll Chlorophyll
Azadirachta 0.9- 0.91- 0.9-
indica Xanthophyll Xanthophyll Xanthophyll
pigment pigment pigment
0.7- 0.6- 0.85-
Chlorophyll Chlorophyll Chlorophyll
Catharanthus 0.78- 0.79- 0.727-
roseus Xanthophyll Xanthophyll Xanthophyll
pigment pigment pigment
Fig-17: Visualization of Catharanthus roseus organic 0.55- - -
fertilizer effected plant pigments under both short and Chlorophyll
long wave lengths of UV

From the experimental research work, we have visualized

various bands of paper chromatography developed by three
different varieties of medicinal plants which were grown
under effect of two different kind fertilizers. Majorly two
different pigments were found to be present in the plant
extracts from the developed chromatograms, and they were
Xanthophylls and chlorophylls [24]. The retention factor also
revealed a great effort for our work, as in the case of
xanthophylls it showed the increase in Rf for the plants treated
with organic fertilizers, and in the case of Chlorophyll
pigment inorganic fertilizer treated plants showed the higher
value when compared to other two different conditions. While
in case of Catharanthus roseus both organic and inorganic
fertilizer treated plants were lacking the chlorophyll pigment

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 324
S.P KIRAN KUMARI* et al.
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY
in the developed chromatogram sheets whereas it was present
in control plant.
5. CONCLUSION
Fertilizers has numerous impact on the plants and as well as in
the environment simultaneously. It gives support to the plants
for building its metabolism up to some extent, at the same
time it exhaust some of its natural and valuable elements
unknowingly .Still those metabolism are to be analyze and
developed to bring awareness in people about the usage of
fertilizers for the growth of plants. The above research work
was the application of biotechnological sciences and it showed
that usage of organic fertilizer may lead to good fruitful results
for the development of these three medicinal plants. In
agricultural circumstances the results may vary from one
tropical region to other, so there is always a need for check it
up by performing some experiments. This work can be further
developed by using more sophisticated equipments like
HPLC, TLC, which may lead to create new innovations in
pharmacological fields.
REFERENCES
[1]. http://www.ask.com/questions-about/Paper-
Chromatography-with-Pigments.
[2]. http://simple.wikipedia.org/wiki/Plant.
[3]. http://nmpb.nic.in/index1.php?linkid=90&level=1&lid=
642
[4].file:///E:/project%20mtech/plant%20project/papers%20coll
ection/medicinal%20plants/Medicinal_plants.htm.
[5]. Glossary of Soil Science Terms". Soil Science Society of
America. Retrieved May 10, 2011.
[6]. M.A Mclean, D.Parkinson. “Changes in structure, organic
matter and microbial activity in pine forest soil following the
introduction of Dendrobaena octaedra (Oligochaeta,
Lumbricidae)”, Dec 1997.
[7]. Glass, Anthony. "Nitrogen Use Efficiency of Crop Plants.
Physiological Constraints upon Nitrogen Absorption". Critical
Reviews in Plant Sciences. Vol 22. No 5. 453. Sep 2003.
[8]. Enwall, Karin, Laurent Philippot, and Sara Hallin.
"Activity and Composition of the Denitrifying Bacterial
Community Respond Differently to Long-Term Fertilization".
Applied and Environmental Microbiology. Vol 71. No 2.pp.
8335–8343. Dec 2005.
IJESAT | Mar-Apr 2012
Available online @ http://www.ijesat.org
ISSN: 2250–3676
Volume-2, Issue-2, 317 – 326
[9]. Lal, R. "Soil Carbon Sequestration Impacts on Global
Climate Change and Food Security". Science. Vol 304. No
5677. pp 1623–7. 2004.
[10]. Rees, Eifion (3). "Change farming to cut CO2 emissions
by 25 per cent". The Ecologist. July 2009
[11]. Fliessbach, A. P Maeder, A Diop, LWM Luttikholt, N
Scialabba, U Niggli, Paul Hepperly, T LaSalle. "Climate
Change: Global Risks, Challenges and Decisions". Earth and
Environmental Science. Vol 6. Feb 2010.
[12]. http://www.ageless.co.za/herb-neem.htm
[13].http://www.flowersofindia.in/catalog/slides/Amaltas.html
,
[14]. http://ntbg.org/plants/plant_details.php?plantid=2497
[15]. http://www.medicinalplants-
flowers.com/2009/06/sadaphuli-catharanthus-roseus.html
[16]. Griffith. Magic and Medicine of Plants: The Reader's
Digest Association. 1993
[17]. Drug Digest: Catharanthus roseus
[18]. Huxley, A. New RHS Dictionary of Gardening.
Macmillan.1992
[19].file:///C:/Documents%20and%20Settings/INSPIRON/Des
ktop/Chromatography.htm
[20]. http://en.wikipedia.org/wiki/Ultraviolet
[21]. http://www.spacewx.com/ISO_solar_standard.html
[22]. http://www.acmasindia.com/blog/uv-trans-illuminator/
[23]. Shoji Shibata, Michio Takito, Osamu Tanaka. “Paper
Chromatography of Anthraquinone Pigments”. J. Am. Chem.
Soc. Vol 72. No 6. pp 2789–2790. 1950
[24]. E Kennedy, H Barker. “Paper Chromatography of
Volatile Acids”. Anal. Chem.Vol 23.No 7. pp 1033–
1034.1951.
325
S.P KIRAN KUMARI* et al. ISSN: 2250–3676
[IJESAT] INTERNATIONAL JOURNAL OF ENGINEERING SCIENCE & ADVANCED TECHNOLOGY Volume-2, Issue-2, 317 – 326

BIOGRAPHIES
S.P Kiran Kumari, M.Tech
Biotechnology, Department of Chemical
Engineering, Andhra University,
Visakhpatnam. Her area of research is
related to Environmental Biotechnology
and molecular biology.

Satya Vani .Yadla M.Tech, Dept. of

Chemical Engineering, Andhra
University ,Visakhpatnam. Her area of
research is Adsorption and Pollution
Control.

Dr.V.Sridevi, Associate professor,

Department of Chemical Engineering,
Andhra university, Visakhapatnam. She
has 12 years of experience in teaching
and has 25 International and National
journals. Her research interests are
biodegradation and environmental
pollution. She has attended 20
conferences and workshops and got an
award of prestigious author from OMICS publications.

Dr. M.V.V Chandana Lakshmi,

Associate professor, Department of
Chemical Engineering, Andhra
university, Visakhapatnam. She has 5
years of experience in teaching and has
25 International and National journals.
Her research interests are
biodegradation and environmental
pollution. She has attended 20
conferences and workshops.

IJESAT | Mar-Apr 2012

Available online @ http://www.ijesat.org 326