Effect of light intensity on the photosynthetic pigment production of Mung bean

plants (Vigna radiata)

Torre, Reyes, Quintans
Lundgren A. Reyes | BIO 120 A-5L

ABSTRACT

Plants are composed of many pigments that aid in the photosynthetic pathways. The production
of these pigments may be affected by many factors including light abundance. In this experiment,
mung bean (Vigna radiata) seedlings were germinated into zero, minimal, and intense light
conditions and then the isolated pigment from the leaves were subjected to paper
chromatography and spectrophotometry to observe the effects of light in pigment production. It
was found out that the dominating plant pigments were chlorophyll a, chlorophyll b, β-carotene,
and lutein. It was also found out that an increase in light intensity also increases the pigment
production in plants since they are very essential for the photosynthetic pathway for the plant to
grow.
should be under a sealed container to prevent
contaminants that might interfere with the
I. INTRODUCTION
results. For plant pigments, dark set-up must be
Plant pigments are chemical compounds employed to prevent the effect of light during
which reflect only certain wavelengths of visible the process of chromatography.
light. These are the reason behind the coloration Since plants are very light dependent.
plants exhibit. They are involved in light energy Under dark conditions, photosynthesis ceases,
gathering processes of photosynthesis which but respiration continues, depleting the
makes them very essential. Chlorophylls are the carbohydrate reserves and triggering a series of
major plant pigments while carotenes are changes in cellular events, like decrease in
termed as accessory plant pigments. Several content of chlorophyll and other plant pigments.
factors such as temperature, pH, and light Spectrophotometry is a good way to compare
intensity greatly affects the overall pigment concentration as well as to observe the
characteristics and function of these pigments. different pigments present or absent on plants
That is why plants in different parts of the world like Vigna radiata subjected to different light
greatly vary in terms of structure and conditions.
characteristics.
II. MATERIALS AND METHODS
Paper chromatography is a technique A. Subjecting Experimental Plants to
that is commonly used to determine the Different Light Intensities
contents of a mixture based on polarity. Sample
solution runs through a specialized paper that is Mung bean seeds were first obtained
highly polar that inhibits the movement of polar and planted on three different pots for the three
substances in the paper, allowing the non-polar different light setups. Each pot setup was
substances to create a trend or line through the allowed to germinate for 12 days on different
paper with the help of a solvent. This process light conditions mainly: complete darkness,
minimal sunlight, and high intensity sunlight.
After germination, the leaves of the mung bean
were cut and placed in three different
containers.
B. Separation of Photosynthetic Pigments
The separation of the photosynthetic
pigments has two major parts. First is the
extraction of photosynthetic pigments and
second is the preparative paper chromatography
and elution of pigments.
For the first part, five grams of fresh
mung bean leaves from the different light setups
were weighed and then washed with distilled
water. For each setup, the leaves were placed in
a pre-cooled mortar and 20ml cold pure acetone
was added. This was done under subdued light
conditions to avoid photobleaching. The leaves
were then homogenized using the pestle. The
filtrate which served as the crude extract was
collected in a 100ml beaker by filtering the
homogenized leaves with the use of a
cheesecloth.
Next, for the preparative paper
chromatography and elution of pigments, 18
strips of approximately ¾" x 6" chromatography
paper were obtained and placed on top of a
clean piece of paper. These were handled using
forceps to avoid fingerprint contamination. Each
chromatography paper was marked 1.5cm from
one end by drawing a light line with a pencil. For
each setup, six chromatography papers were
used, and prepared crude extract were applied
carefully and evenly along the lines using a
capillary tube and were dried after. The
application with drying in between was repeated
approximately 20 times until the line turned dark
green and the final thickness of the streak was
no more than 6-7mm. As the strips were drying,
the developing set-up was prepared, consisting
of a 250ml beaker wrapped and covered with
carbon paper which contains 20ml of 9:1
petroleum-ether: acetone. This was executed
with caution since the solvent is highly
flammable, and inhalation of fumes were
avoided. Using forceps, the top end of each strip
(without the extract) was handled and the
bottom edge (with extract) was carefully
lowered towards the developing solution. The
same procedure was done for the other strips
were spaced evenly along the side of the beaker.
The beaker was then covered with carbon paper.
The chromatogram development was allowed to
occur until the solvent has reached about 0.5-
1cm from the top of the paper. After
development, the chromatograms were
removed and air dried. Using forceps to handle
the strips, the bands from all the strips were cut
and grouped according to color. Corresponding
to the number of colors obtained, the same
number of tubes were prepared, each tube
containing 4ml of pure acetone. To obtain
partially purified pigments, the pigments were
eluted by putting one group of colored bands in
one tube and shaking the tubes for five to ten
minutes.
C. Spectrophotometric Characterization
of Pigments: Determination of the
Absorption Spectra
The blank consisting of pure acetone
was placed in a cuvette. Each partially purified
pigment from each mung bean sample was
transferred to a separate cuvette to leave out
the thin strips from which the pigments were
eluted. The absorbance of each sample from
350nm to 700nm at 25nm intervals were read
and the absorbance against wavelength was
plotted. The obtained spectra were compared
with the known spectra of the colored pigments
found in references. From these, the pigments
separated were identified.
[AUTHOR NAME] 2
 III. RESULTS AND DISCUSSIONS

0.08
0.07
0.06
0.05
Absorbance

0.04
0.03
0.02
0.01
0
-0.01 400 450 500 550 600 650
Wavelength (nm)

Pigment 1 Pigment 2 Pigment 3 Pigment 4

Figure 1. Absorption spectra of different photosynthetic pigments obtained from Mung bean plants
(Vigna radiata) subjected to no amount of light.

Figure 2. Representative chromatogram of different photosynthetic pigments obtained from Mung

bean plants (Vigna radiata) subjected to no amount of light.

A. Mung Bean Exposed in Darkness
For the mung bean leaves subjected to full
darkness, four different pigments were seen in
the chromatogram as shown in Figure 2. It can
be observed that pigments 1 and 2 are more
evident than pigments 3 and 4. The figure also
shows that pigment 1 is colored dark yellow,
pigment 2 is colored yellow while pigment 3 is
dark green, and pigment 4 is light green. Figure
1. shows the absorption spectra of the four
pigments isolated. Pigment 1 peaks at around
425-450 nm., pigment 2 peaks at 425 nm.,
pigment 3 at 425nm. and 675nm. and lastly,
pigment 4 at 425nm. and 650nm.
According to Lee (2007) the four
common plant pigments are chlorophyll a,
chlorophyll b, xanthophyll (lutein), and
carotene(β-carotene). In acetone, chlorophyll a

[AUTHOR NAME] 3
has a maximum absorbance at 430nm and
662nm and is usually dark green in appearance.
Chlorophyll b on the other hand has a maximum
absorbance at 455nm and 645nm and is usually
light green. β-carotene absorption peaks at
452nm and is usually yellow orange in color
while lutein peaks at 445nm and is usually yellow
in color (Bulda, O., Rassadina, V., Smolikova, G.,
& Laman, N. (2008). Lastly, in an experiment by
Stovall (2013) the chromatogram shows that the
pigments are arranged as carotenoid,
xanthophyll, chlorophyll b, and chlorophyll b
from top to bottom. Comparing these to the
gathered data, can help determine the probable
identity of the isolated pigments. I
Pigment 1 which is dark yellow in color
and has peak absorbance at 425-450nm has
similarity with β-carotene which peaks at 452nm
and is yellow orange in color. Both carotene and
pigment 1 are also found at the topmost of the
chromatogram. This may mean that pigment 1 is
β-carotene or of the carotene group. Pigment 2
which is yellow and has an absorbance peak at
425nm can be compared to lutein since it is also
yellow and has an absorbance peak at 455nm
which is relatively near. Both are also the second
(from the top) pigment in their chromatograms,
so this may tell that pigment 2 is lutein or of the
xanthophyll group. Pigment 3 is very similar to
chlorophyll a since their peak absorbances are
very near (425nm/675nm and 430nm/662nm
respectively). Also, pigment 3 and chlorophyll a
are both dark green in color and are the third
(from the top) pigment in their chromatograms.
This may mean that pigment 3 is chlorophyll b or
of the chlorophyll group. Pigment 4 on the other
hand is very similar to chlorophyll b since their
peak absorbances are also near (425nm/650nm
and 455nm/645nm). Also, pigment 4 and
chlorophyll b are both light green in color and are
the fourth (from the top) pigments in their
respective chromatograms.
B. Mung Bean Exposed in Minimal
Sunlight
For the mung bean leaves subjected to
minimal sunlight, only three distinguishable
pigments were seen in the chromatogram as
shown in Figure 4. It can be observed that
pigments 2 and 3 are more evident than pigment
1. The figure also shows that pigment 1 is colored
faint yellow, pigment 2 is dark green, and
pigment 3 is light green. Figure 3. shows the
absorption spectra of the three pigments
isolated. Pigment 1 peaks at around 425-450nm
and 650nm, pigment 2 peaks at 425nm and
650nm, and pigment 3 peaks at 425nm.
Again, using the absorbance values
observed by Bulda et al. (2008) and
chromatogram by Stovall (2013), it can be
observed that only pigment 2 coincides with
literary pigment observations and values
because the gathered absorbances in this part of
the experiment were fluctuating. The most
probable identity of pigment 2 is chlorophyll a
since their peak absorbances are very near
(430nm/662nm for chlla and 425nm/650nm for
pigment 2). Also, pigment 2 and chlorophyll a are
both dark green in color and are the second
(from the bottom) pigment in their
chromatograms. For pigment 1, the two peak
absorbances do not coincide with its position
and color in the chromatograms compared to
literary values. For assumption purposes,
pigment 1 may be of the carotenoid group
(either carotene on xanthophyll) because of its
position as the first pigment (from the top) of the
chromatogram as well as its dark yellow color.
For pigment 3, the single peak absorbances do
not coincide with its position and color in the
chromatograms as well as peak absorbances
from literary values. For assumption purposes,
pigment 3 may be of the chlorophyll group, most
probably chlorophyll b because of its position as
[AUTHOR NAME] 4
the first pigment (from the bottom) of the
chromatogram as well as its light green color.

0.14
0.12
0.1
Absorbance

0.08
0.06
0.04
0.02
0
-0.02 425 475 525 575 625 675
Wavelength (nm)

Pigment 1 Pigment 2 Pigment 3

Figure 3. Absorption spectra of different photosynthetic pigments obtained from Mung bean plants
(Vigna radiata) subjected to minimal sunlight.

Figure 4. Representative chromatogram of different photosynthetic pigments obtained from Mung

bean plants (Vigna radiata) subjected to minimal sunlight.

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 2.5
1.5
Absorbance
0.5
0
400 450 500
-0.5
Wavelength (nm)
Pigment 1
Figure 5. Absorption spectra of different photosynthetic pigments obtained from Mung bean plants
(Vigna radiata) subjected to intense sunlight.
C. Mung Bean Exposed in Intense
Sunlight
For the mung bean leaves subjected to
intense sunlight, only three distinguishable
pigments were seen in the chromatogram.
Unfortunately, due to human error no picture of
the chromatogram was recorded. It was
observed that pigments 1, 2 and 3 are very
evident. It was also observed that pigment 1 is
colored yellow, pigment 2 is dark green, and
pigment 3 is light green. Figure 5. shows the
absorption spectra of the three pigments
isolated. Pigment 1 peaks at around 425-450nm,
pigment 2 peaks at 425nm and 650nm, and
pigment 3 peaks at 425nm and 650-675nm.
By using the absorbance values
observed by Bulda et al. (2008) and
chromatogram by Stovall (2013), the most
probable identity of pigment 2 is chlorophyll a
since their peak absorbances are very near
(430nm/662nm for chlla and 425nm/650nm for
550 600 650
Pigment 2 Pigment 3
pigment 2). Also, pigment 2 and chlorophyll a are
both dark green in color and are the second
(from the bottom) pigment in their
chromatograms. The most probable identity of
pigment 3 on the other hand, is chlorophyll b
since their peak absorbances are also near
(455nm/645nm for chllb and 500nm/650nm for
pigment 2). Also, pigment 2 and chlorophyll a are
both light green in color and are the first (from
the bottom) pigment in their chromatograms.
Lastly, pigment 1 may be of the xanthophyll
group (lutein) because of its position as the first
pigment (from the top) of the chromatogram as
well as its yellow color. Also, Pigment 1’s peak
absorbance (425nm) is closer to lutein’s (445nm)
than β-carotene’s (452nm).
D. Effect of Different Light Intensities on
Pigment Production
Along with the determination of the
photosynthetic pigments present in mung bean
leaves subjected to different light conditions is
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the assessment of the effects of these light
conditions in pigment production. First, it was
observed through the chromatograms that on
the mung bean under darkness, two faint bands
chlorophyll and two distinct bands of
carotenoids were seen. Under minimal sunlight,
two distinct bands of chlorophyll, and a faint
band of carotenoid developed. Under intense
sunlight, two very distinct bands of chlorophyll
and a very distinct band of carotenoid can be
seen. It can also be observed through
comparison of figures 1,3, and 5 that there is an
overall increase in absorbance values of the
different pigments as the amount of light
increased. According to Lambert-Beer law, there
is a linear relationship between absorbance and
concentration (Swinehart, 1963). These
observations indicate there has been an increase
in overall pigment concentration (more so on the
chlorophyll content compared to carotenoid
content) as the amount of light increased.
De-etiolation is a series of physiological
and biochemical changes a plant shoot
undergoes when emerging from the ground or in
response to light after a period of insufficient
light exposure. This process is known informally
as greening. During greening, the plant starts to
increase the production of pigments to harvest
more light that will power the biosynthetic
pathways. Under complete darkness, the
etioplasts, which are chloroplasts with inactive
photosynthetic pigments dominate the leaves
(Reece & Campbell, 2011). This explains the faint
bands of chlorophyll as well as low absorbance
values observed under the first setup. When
plants are subjected to sunlight, the transition of
etioplasts to chloroplasts is accompanied by the
production of major and antenna or accessory
plant pigments (Wise, 2007). Major plant
pigments capture the energy of sunlight and has
a direct role in the photosynthetic pathway while
accessory pigments cannot transfer sunlight
energy directly to the photosynthetic pathway
but must pass their absorbed energy to
chlorophyll (Mulder-Krieger & Verpoorte, 1994).
This may explain the relative abundance of
chlorophyll a and b, which are major plant
pigments compared to lutein and β-carotene
which are accessory plant pigments as the plant
is subjected to higher light intensity.
Overall, it can be said that the increase
in plant pigment production is light dependent
and is very essential for the growing plant.
E. Errors in Spectrophotometry
It can be observed that a lot of the values
gathered from spectrophotometry are
inconsistent and fluctuation with respect to
literary values. Also, the peak absorbances were
not the same and only the closeness of the
values was considered. The accuracy of the
obtained results depends on the contamination
of the extracts with, for instance, precursors and
products of chlorophyll catabolism, which
spectra overlap with those of chlorophylls (Bulda
et al.,2008)). Contamination during elution may
also cause this. Moreover, the low pigment
concentration challenges the capability of the
spectrophotometer in detecting low amounts of
absorbance values (thus yielding some negative
absorbance values). It is suggested that for
further experiments, proper handling of samples
should be exercised and as well as a more
advanced spectrophotometer can be used. An
increase in initial concentration of the samples
are also encouraged.
IV. LITERATURE CITED
Bulda, O., Rassadina, V., Smolikova, G., & Laman,
N. (2008). Spectrophotometric
measurement of carotenes,
xanthophylls, and chlorophylls in
extracts from plant seeds. Russian
Journal of Plant Physiology. 55. 544-551.
10.1134/S1021443708040171.
[AUTHOR NAME] 7
Lee, D (2007). Nature's palette - the science of
plant color. University of Chicago Press
Mulder-Krieger T., Verpoorte R. (1994) Plant
Pigments. In: Anthocyanins as Flower
Pigments. Springer, Dordrecht
Reece, J. B., & Campbell, N. A. (2011). Campbell
biology. Boston: Benjamin Cummings /
Pearson.
V. APPENDIX
Table 1. Positions of maximum and specific
absorption coefficients (α) of chlorophyll a and
b, β-carotene, lutein in acetone and in the
mixture of petroleum ether and
tetrahydrofuran by Bulda et al. (2007).

Stovall, G. (2013). Spinach Leaf Pigments.

Retrieved May 16, 2018 from
http://pulpbits.net/6-leaf-
chromatography-pictures/spinach-leaf-
pigments/

Swinehart, D. (1962). The Beer-Lambert Law. J.

Chem. Educ., 1962, 39 (7), p 333.
10.1021/ed039p333

Wise R.R. (2007). The Diversity of Plastid Form

and Function. In: Wise R.R., Hoober J.K.
(eds) The Structure and Function of
Plastids. Advances in Photosynthesis and
Respiration, vol 23. Springer, Dordrecht

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 Table 3. absorbance values of different
photosynthetic pigments obtained from Mung
bean plants (Vigna radiata) subjected to
minimal sunlight.
Absorbance
Wavelength (nm)
Pigment 3 Pigment 2 Pigment 1
425 0.129 0.092 0.008
450 0.109 0.025 0.006
475 0.037 0.027 0.001
500 0.008 0.001 -0.003
525 0.009 0.005 -0.004
550 0.007 0.012 -0.003
575 0.013 0.008 -0.003
600 0.023 0.014 -0.003
625 0.025 0.015 -0.006
650 0.06 0.04 -0.003
675 0.026 0.033 -0.003

Figure 1. Sample chromatogram of spinach leaf

pigments by Stoval (2013). Table 4. absorbance values of different
photosynthetic pigments obtained from Mung
Table 2. absorbance values of different bean plants (Vigna radiata) subjected to
photosynthetic pigments obtained from Mung intense sunlight.
bean plants (Vigna radiata) subjected to
Absorbance
complete darkness. Wavelength (nm)
Pigment 3 Pigment 2 Pigment 1
Absorbance 400 0.045 0.618 0.182
Wavelength
(nm) Pigment Pigment Pigment Pigment 425 0.095 1.999 0.484
4 3 2 1
450 0.074 0.52 0.474
400 0.04 0.041 0.033 0.024
475 0.061 0.031 0.252
425 0.048 0.047 0.031 0.028
500 0.008 0.186 1.999
450 0.047 0.035 0.008 0.013
525 -0.002 0.179 0.48
475 0.028 0.016 0 0.003 550 -0.003 0.176 0.59
500 0.01 0.002 -0.001 -0.002 575 0 0.217 0.075
525 0.001 0.001 -0.001 -0.002 600 0.002 0.251 0.083
550 -0.001 0.001 0 -0.002 625 0.001 0.282 0.085

575 0.002 -0.001 0.002 0.002 650 0.011 0.524 0.179

675 0.01 0.334 0.055
600 0 -0.002 0.004 0.001
625 -0.001 -0.003 0.001 0
650 0.003 0.001 0.01 0.07
675 0.003 0.001 0.047 0.006

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