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Using Chromatography to Separate Plant Pigments

November 15, 2016
Dilpreet Matharu
791237
Ms
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A piece of paper was
cut carefully, preventing it from touching the bottom of the test tube
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5-10 mL of
solvent was added in the test tube
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Data was collected by measuring the distance that each pigment
travelled and by calculating the Rf values
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Higher the reference values, non- polar the pigment and vice-versa
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Introduction:
Chromatography, developed by Michail Tswett, is a simple yet effective
technique used by scientists for the separation of a mixture by passing it in solution or
suspension through a medium for the individual components to be thoroughly analyzed
...
) This technique is used to
examine the pigments in a green leaf
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d
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Pants nature’s food factories are multicerellular eukaryotes of the kingdom
Plantae
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Photosynthesis enables plants to produce structural and
metabolic substances that aid in their survival (McGraw 2011)
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It is represented by the equation 6Co2 + H2O  C6H12O6 + 6O2
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They are called the lightdependent reactions and the light independent reactions (McGraw 2011)
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Solar energy that is converted into glucose is a form of energy that can
travel in wave packets called photons, which has a fixed quantity of energy
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It
is also depicted as the degree of brightness that a plant is exposed to (Bareja, 2011)
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In plants, the carbon dioxide first enters
through openings, called stomata, in the leaves
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Thylakoids are saclike membranes within the chloroplasts,
containing the molecules that absorb energy from sun
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The green colour of spinach is due to it containing more green
chlorophyll, which dominates the other pigments to give the overall green colour
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It is noticeable from the

investigations that only the red and blue ends of the visible part of the electromagnetic
spectrum are used by plants in photosynthesis and transmit or reflect green light (“Light
absorption for photosynthesis” n
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Figure 1: Demonstrates the
absorbance spectrum when
accessory pigments are combined
with chlorophyll a and chlorophyll b
(McGraw Hill 2011)
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A chlorophyll solution absorbs red and blue light, and it transmits green light (Mcgraw
2011)
...
Additionally, an
action spectrum (figure 4) demonstrated the relative effectiveness of different
wavelengths of light for promoting the process of photosynthesis
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Figure 4: This demonstrates the action
spectrum for photosynthesis, it shows the
rate at which O2 is produced when different
wavelengths of light are shown on the leaf
(McGraw Hill 2011)
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Accessory pigments pass
their absorbed energy onto photosystem
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When chlorophyll molecules are associated with different
proteins in a process called photosystem, they are capable of absorbing light energy of
various wavelengths (McGraw 2011)
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ATP and NADPH are then used to incorporate carbon dioxide into organic
molecules through a process called carbon fixation (“Plant Pigments and
Photosynthesis Lab Report”, n
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Although, there are many methods for separating the green pigment,
chromatography is the most efficient technique
...
Isopropanol is used as a solvent instead
of water because it is less polar than water
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This concludes, larger the alkyl group, better the
solvent (Bett, 2014)
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The mobile phase
solvent will rise slowly up the paper by capillary action
...
The
separation of pigments and the distance covered by these pigments will depend on the
type of leaf and the type of solvent
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Since, the spinach leaf is green,
the chromatograph will mainly have chlorophyll A and B
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Materials
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Spinach leaves



Isopropanol (solvent)

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Chromatography paper



Paper clip



Retort stand or test tube rack



Test- tube clamp



Cork stopper



Pipette



Large test tube



Pencil

Experimental Procedure:
The procedure of this lab was obtained from McGraw Hill, 2011 on page number 176
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Experimental Observations:

Chlorophyll b (yellowish-green)

Figure 5: This chromatograph was obtained from the lab
conducted in the class
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The lab was not successful as
more pigments were expected to be seen on the chromatograph
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This was achieved by analyzing the chromatograph and
calculating the distance travelled by solvent and pigment
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3

3
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3

2
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3

6
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3

4
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4 cm
6
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38

Rf value for chlorophyll a: 0
...
73
Rf value for xanthophyll: 0
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The pigment called
carotenoid was the most soluble
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The results were obtained by looking at what pigment travelled farthest
...
d
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While comparing the observations and Rf values with the classmates, it
was noticed that there could be possible indeterminate errors
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In this lab, some possible indeterminate errors that can affect the data are
contamination of chemicals used and properties of the mixture of spinach leaves
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Another possible
error could be caused by not keeping a check of relative humidity
...
d
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The
outcome of the chromatograph showed four different pigments
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The dominating pigment was green even though it contained other pigments because it
is made up of more green chlorophyll, which overpowers the other pigments to give the
overall green colour
...
This could be tested
by running the chromatography in different solvents to get additional pigments
...
It was understood that
the spinach leaf not only carries chlorophyll a but also carries other pigments such as
carotenoids, xanthophyll and chlorophyll b through the chromatography
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It was discovered that
the chromatograph done in class was not successful due to possible indeterminate
factors
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References
Academic Writing Service - WriteMyPapers
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(n
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Retrieved from
http://www
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org/examples-and-samples/paper-chromatographyexperiment-report
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(n
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Retrieved from
http://study
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html
Bett, S
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Organic Chemistry: When would one use isopropanol as a
solvent rather than water? Retrieved from https://www
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com/OrganicChemistry-When-would-one-use-isopropanol-as-a-solvent-rather-than-water
Bullard, J
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McGraw-Hill Ryerson biology
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Chromatography Lab Answers| Online Homework Help | SchoolWorkHelper
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d
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net/chromatography-lab-answers/
Chromatography
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d
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blogspot
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html
DiGiuseppe, M
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(2012)
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Scarborough, ON: Nelson Thomson Learning
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(n
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Chromatography
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ibsurvival
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(2014, January 29)
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education
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(n
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Retrieved from http://hyperphysics
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gsu
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html
Light Intensity in Plant Growth and Development
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d
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cropsreview
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html
Paper Chromatography Lab
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Retrieved from
https://prezi
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(n
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doi:http://www
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edu/wp/chelifossum/files/2011/08/Exp-3-paper-chrom-30A376
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(n
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Retrieved from https://www
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org/testprep/mcat/chemical-processes/separations-purifications/a/principles-ofchromatography
Stubbings, J
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d
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Retrieved from
http://www
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com
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html

University, A
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(n
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Paper Chromatography
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olabs
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in/?sub=79&brch=17&sim=124&cnt=1
University Follow, A
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Paper chromatography experiment
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slideshare
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d
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reference
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d
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reference

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