Activity 5

Laboratory nstructor: Ms.

Olgga A. Hara
Post Lab. Discussion by:
Group 1
Lozano, Tweela
Magpantay, Marielle
Mendigorin, Kristine
Mercado, Jerra
Miyata, Michiko
W An organelle found within the cells of green
plants and eukaryotic algae which contains the
membranes, photosynthetic pigments, and
enzymes necessary for photosynthesis.
< A pigment that is present in chloroplasts that
captures the light energy necessary
for photosynthesis.
< Pigments are "molecules that absorb specific
wavelengths (energies) of light and reflect all
others."
< Any light that does not have enough or has too
much energy can not be absorbed and is
reflected.
< Green plants have six closely-related
photosynthetic pigments (in order of increasing
polarity):
4 ,7otene - ,n o7,nge pigment
4 X,nthophyII - , yeIIow pigment
4 hIo7ophyII , - , bIue-g7een pigment
4 hIo7ophyII - - , yeIIow-g7een pigment
< hlorophyll a is the most common of the six,
present in every plant that performs
photosynthesis.
< The reason that there are so many pigments is
that each absorbs light more efficiently in a
different part of the spectrum.
< hlorophyll a - 400-450 nm and at 650-700 nm
< hlorophyll b - 450-500 nm and at 600-650 nm.
< Xanthophyll - 400-530 nm.
< However, none of the pigments absorbs well in the
green-yellow region, which is responsible for the
abundant green we see in nature.
`Tuba-tuba
(,9745, .:7.,8
L.) leaves used as
sample (group 1)
Leaves washed with distilled water and
were cut into small pieces.
Leaves were placed in a pre-cooled mortar with 20ml cold pure
acetone used as a solvent.
Leaves were grinded using the pestle in subdued lighting.
ote: Conducting the activity in subdued lighting is
needed to prevent pigment degradation.
Chlorophyll oI plants are extremely light
sensitive and easily destroyed by photobleaching
or exposure to light. They are also Iragile and
very unstable.
Light, in the presence of molecular oxygen (O
2
),
degrades pigments (photo-oxidation)
Homogenate was filtered using cheesecloth. The collected filtrate was placed
in a 100ml beaker.
Green Suspension was transferred to a cold 50ml centrifuge tube. t was
centrifuged at 1500 rpm for 1 minute at 4.
*Filtration is done to remove large debris (e.g., cell walls),
unbroken cells and fragments.
* The low-speed centrifugation will sediment remaining large
bodies from the filtrate.
Supernatant was decanted into a clean centrifuge tube.
The remaining suspension was recentrifuged. But now in 4000 rpm for 10
minutes.
*Moderate-speed centrifugation will sediment chloroplasts, leaving
mitochondria and ribosomes and soluble components in the
supernatant.
Supernatant was discarded.
hloroplast pellet was resuspended in 5.0 ml of cold 0.35 M Nal.
Vortex was used to disrupt the packed pellet.
hloroplast suspension was observed in microscope (OO)
Mark a llghL llne on each chromaLography paper aL 13 cm from
Lhe boLLom end
Carefully and evenly apply Lhe crude chlorophyll suspenslon
uslng caplllary Lube along Lhe llne Allow Lo dry 8epeaL Lhe
appllcaLlon wlLh drylng 10x unLll Lhe llne Lurns dark green
Obtain 5 strips of 2x15 chromatography paper
Jrap Lhe LesL Lubes wlLh alumlnum foll our 20mL of 91 peLroleum
eLher aceLone lnLo Lhe LesL Lube (uLvLLClnC SL1u)
Carefully lower Lhe boLLom edge (wlLh exLracL) Lowards Lhe
developlng soluLlon lnslde Lhe Lube
Allow Lhe chromaLogram developmenL Lo occur
unLll Lhe solvenL has reached 031 cm from Lhe
Lop of Lhe paper
P,pe7 h7om,tog7,phy
W hromatography is a technique used to separate the components of
a mixture. There are various types of chromatography (column,
paper, thin-layer, gas).
W but in all cases the separation is achieved by distribution of
components between a fixed or stationary phase (Filter Paper) and
a moving or mobile phase (Solvent) by polarity of the substances
used.
n paper chromatography, the
components of a mixture are
separable into discrete zones on a
sheet of filter paper.
With a capillary tube, the mixture is streaked on the
chromatography paper: enough sample is applied so
that there will be an adequate amount for subsequent
extraction and spectrophotometric analysis.
rude chlorophyll
suspension applied on the
chromatogram
ellow, a lighter yellow, and an
almost clear bands of pigments
were formed. These bands of
pigments were not readily seen in
our sample leaves. Only green
coloration of the leaves was
observed.
Bands of Pigments Produced:
ellow
Light yellow
Light yellow
(almost clear)
The bands of pigment formed in the chromatogram are
cut and then grouped according to color.
n separate tubes, 4 mL
of pure acetone was
added.
Each group of
pigments were
added to each test
tubes. The tubes
were shaken for 10
minutes.
&sing a transfer pipette, eluted
epigments were transferred to a
cuvette.
To another cuvette, pure
acetone was added.
Absorbance was read from 350
to 700 nm (at 25 nm interval)
Absorbance vs. avelength
was plotted
Consists oI two instruments:
spectrometer Ior producing light oI any selected color
(wavelength), and a photometer Ior measuring the intensity oI light
The instruments are arranged so that
liquid in a cuvette can be placed
between the spectrometer beam and the
photometer. The amount oI light
passing through the tube is measured by
the photometer.
Spectrophotometer
(photometer) (spectrometer)
display
The colours displayed by
different pigments are
result of the light that is
being reflected or
transmitted by the
molecules.
The wavelength of
light that is most
ABSORBED would be
complimentary to the
wavelength that is best
TRANSMITTED .
They would be found
on opposite sides of
the &# WHEE.
Thus, a green pigment would be
expected to show the greatest
ABSORBANE in the red region of
the spectrum (640 700 nm).
Spectrophotometer in calculation of pigment
concentration:
Pigment concentrations are calculated by taking the absorbance
reading of the highest peak in a pigment spectrum, as characterized by
a spectrophotometer, and dividing that absorbance value by the
pigment's specific absorption or molar absorption coefficients.
Specific Absorption oefficient (L g-1 cm-1)
oncentration of
Pigment (mg/)

Absorbance &nits (Au)

x 1000
*SPEF ABSORPTON OEFFENT
A standard universal value (for each pigment) usually given in
volume per weight with respect to path length of the cuvette used.
-0.02
-0.01
0
0.01
0.02
0.03
0.04
0.05
0 100 200 300 400 500 600 700 800
Pigment 1
W,;eIength ;s. Abso7b,nce
Absorbance of pigment: at
00 00- -500 nm 500 nm
1. PGMENT ELLO
Possible dentity of the
Pigment: Xanthophyll Xanthophyll
WXanthophyll is a yellow pigment from the
carotenoid group. Xanthophylls absorb well at a
wavelength of 400-530nm.
WThere are so many pigments present in
chlorophyll because each of them absorbs light
more efficiently in a different part of the spectrum.
WHowever in the experiment, only yellow pigment
(xanthophyll) was observed in chromatogram.
-0.25
-0.2
-0.15
-0.1
-0.05
0
0.05
0 100 200 300 400 500 600 700 800
Pigment 2
W,;eIength ;s. Abso7b,nce
2. PGMENT LGHT ELLO
Absorbance of pigment: at
50 50- -700 nm 700 nm
-0.02
-0.015
-0.01
-0.005
0
0.005
0.01
0.015
0.02
0 100 200 300 400 500 600 700 800
Pigment 3
W,;eIength ;s. Abso7b,nce
3. PGMENT LGHT ELLO
TO ALMOST LEAR
Absorbance of pigment: at
50 50- -700 nm 700 nm
Possible dentity of Pigments 2 & 3: nthoxanthin nthoxanthin
WAnthox,nthins are a type of flavonoid pigments
in plants. Anthoxanthins are water-soluble
pigments which range in color from white or
colorless to a creamy to yellow, often on petals of
flowers.
WThese pigments are very susceptible to color
changes. Thus, explains why the leaves of
Jatropha curcas are still green even though
Anthoxanthin is generally present.
< hloroplast. The Encyclopedia of Science. Available from: http://www.
daviddarling.info/encyclopedia//chloroplasts.html.
< Parts of hloroplasts. Retrieved on 3 Aug 2011 from http://micro.magnet.fsu.edu/
cells/chloroplasts/chloroplasts.html.
< Photosynthetic Pigments. Retrieved on 3 Aug 2011 from http://www.ucmp
.berkeley.edu/glossary/gloss3/pigments.html.
< Jatropha curcas L. image. Retrieved 3 August 2011 from http://chalk.richmond.edu/flora-
kaxil-kiuic/j/Jatropha_curcas_&_02s.JPG
< Separation of plant pigments by paper chromatography. Available from
http://www.chemtopics.com/unit0/pchrom.pdf
< Photosynthetic pigments. Retrieved 3 Aug 2011 from
http://www.tutorvista.com/content/biology/biology-iv/photosynthesis/photosynthetic-
pigments.php
< Action and absorption spectra. Available from
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages//ctionSpectrum.html
< Absorbance application notes: pigment concentration. Retrieved 5 August 2011 from
http://www.turnerdesigns.com/t2/doc/appnotes/S-0075.pdf
Pet7oIeum Ethe7 will serve as the
polar part of the solution
Acetonewill serve as the non-polar
part of the solution
Remember: "Like dissolves like
Photosynthetic pigments are composed of polar
and non-polar substances. Therefore, polar and
non-polar solvents should also be used so that all
components of the pigments can travel the
chromatogram.