The effect of light intensity on the amount of chlorophyll in Cicer arietinum



                               Extended Essay

                                Biology (SL)



    The effect of light intensity on the amount of chlorophyll in Cicer
                                 arietinum



                                                     Word count: 4 413 words


Content



Abstract  2
Introduction .. 3
Hypothesis  3
Method:
Description .... 8
Results .. 10
Discussion .... 14
Conclusion .... 14
Evaluation of the method .. 15
Bibliography . 16
                                  Abstract.
      Plants, growing on the shaded area has less concentrated  green  color
and are much longer and thinner than plants growing  on  the  sun  areas  as
they are dark green, short and thick. Research question was: How  does  the
amount of chlorophyll-a and chlorophyll-b, gram per gram of  plant,  depends
on the light intensity in which plants are placed?
      Hypothesis suggests that there are several  inner  and  outer  factors
that affect the amount of chlorophylls a and b in plants and that  with  the
increase of light intensity the amount of  chlorophyll  will  also  increase
until light intensity exceeds  the  value  when  the  amount  of  destructed
chlorophylls is greater than formatted thus decreasing the total  amount  of
chlorophylls in a plant.
      The seeds of Cicer  arietinum  were  divided  into  seven  groups  and
placed into various places  with  different  values  of  light  intensities.
Light intensities were measured with digital colorimeter. After three  weeks
length was measured. Then plants were cut and quickly dried.  Their  biomass
was also measured. Three  plants  from  each  group  were  grinded  and  the
ethanol extract of pigments was prepared. The  amount  of  chlorophylls  was
measured using method of titration and different formulas.
      The investigation showed that  plants  growing  on  the  lowest  light
intensity equal 0 lux contained no chlorophyll and had the  longest  length.
The amount of chlorophyll quickly increased and length  decreased  with  the
increase of  light  intensity  from  0  lux  to  1200  lux.  The  amount  of
chlorophyll in plants unpredictably decreased during light  intensity  equal
to 142 lux  and  than  continued  increasing  and  didnt  start  decreasing
reaching very high value (1200 lux).
      The sudden decrease happened due to mighty  existence  of  some  inner
genetical damages of seeds which  prevented  them  from  normal  chlorophyll
synthesis and predicted decrease  didnt  decrease  because  extremely  high
light intensity was not exceeded.
                                                       Word count: 300 words



                              I. Introduction.
      This theme seemed to be attractive for me because  I  could  see  that
results of my investigation could find application in real life.
      While walking in the  forest  in  summer  I  saw  lots  of  plants  of
different shades of green color: some of them were  dark  green,  some  were
light green and some even very-very light green with yellow shades, hence  I
became very interested in this situation and wanted to know why  it  happens
to be so. I also saw that those plants that were growing on sunny  parts  of
forest, where trees were not very high, had  dark  green  color  and  those,
that were growing in shady parts of the same forest  had  very  light  green
color. They also had difference in their length and thickness  those,  that
were growing on light were very short, but  thick  and  strong,  and  those,
growing in shady regions were very thin and fragile.
      Hence I became very interested in finding scientifical description  of
 my observations.
      The aim of my project is to find out how does  the  changes  in  light
intensity affect balance of chlorophyll in Cicer arietinum.

                               II. Hypothesis.


      There are several factors that affect the development  of  chlorophyll
in plants.[1]
      Inner factors. The most important one is  genetical  potential  of  a
plant, because sometimes  happen  mutations  that  follow  in  inability  of
chlorophyll formation. But most of the times it happens that the process  of
chlorophyll synthesis  is  broken  only  partly,  revealing  in  absence  of
chlorophyll only in several parts of the plant or in  general  low  rate  of
chlorophyll.  Therefore  plants  obtain  yellowish   color.  Lots  of  genes
participate in the process of  chlorophyll  synthesis,  therefore  different
anomalies are widely represented. Development  of  chloroplasts  depends  on
nuclear  and  plastid  DNA  and  also  on  cytoplasmatic  and  chloroplastic
ribosomes.
      Full provision of carbohydrates seem to be essential  for  chlorophyll
formation,  and  those  plants  that  suffer   from   deficit   of   soluble
carbohydrates may  not  become  green  even  if  all  other  conditions  are
perfect. Such leaves, placed into sugar  solution  normally  start  to  form
chlorophyll.  Very  often  it  happens  that   different   viruses   prevent
chlorophyll formation, causing yellow color of leaves.
   Outside factors.  The  most  important  outside  factors,  affecting  the
formation of chlorophyll are: light  intensity,  temperature,  pH  of  soil,
provision of minerals, water and oxygen. Synthesis of  chlorophyll  is  very
sensitive to all the factors, disturbing metabolic processes in plants.
   Light. Light is very important for the chlorophyll formation, though some
plants are able to produce chlorophyll in absolute darkness. Relatively  low
light intensity is rather  effective  for  initialization  and  speeding  of
chlorophyll development. Green plants grown in darkness  have  yellow  color
and contain protochlorophyll  predecessor of  chlorophyll  ,  which  needs
lite to restore until chlorophyll . Very high light  intensity  causes  the
destruction of chlorophyll. Hence chlorophyll is synthesized and  destructed
both at the same time.  In  the  condition  of  very  high  light  intensity
balance is set during lower chlorophyll concentration, than in condition  of
low light intensity.
   Temperature. Chlorophyll synthesis seems to happen  during  rather  broad
temperature  intervals.  Lots  of  plants  of        synthesize
chlorophyll from very low temperatures till very high  temperatures  in  the
mid of the summer. Many pine trees loose  some  chlorophyll  during  winters
and therefore loose some of their green color. It  may  happen  because  the
destruction  of  chlorophyll  exceeds  its   formation   during   very   low
temperatures.
   Provision with minerals. One of the most common reason  for  shortage  of
chlorophyll is absence of some  important  chemical  elements.  Shortage  of
nitrogen is the most common reason for lack of chlorophyll  in  old  leaves.
Another one is shortage of ferrum, mostly in young leaves  and  plants.  And
ferrum is important element for chlorophyll synthesis. And  magnesium  is  a
component of chlorophyll therefore its shortage causes  lack  of  production
of chlorophyll.
   Water. Relatively low water stress lowers speed of chlorophyll  synthesis
and high dehydration of  plants  tissues  not  only  disturbs  synthesis  of
chlorophyll, but even causes destruction of already existing molecules.
       Oxygen.  With the absence of oxygen plants do not produce
chlorophyll even on high light intensity.  This shows that aerobic
respiration is essential for chlorophyll synthesis.

      Chlorophyll.[2] The synthesis of  chlorophyll  is  induced  by  light.
With light, a gene can be transcripted and translated in a protein.
The plants are naturally blocked in the  conversion  of  protochlorophyllide
to chlorophyllide. In normal plants  these  results  in  accumulation  of  a
small  amount  of  protochlorophyllide  which  is  attached  to   holochrome
protein. In vivo at least two types of  protochlorophyllide  holochrome  are
present. One, absorbing maximally at approximately 650  nm,  is  immediately
convertible to chlorophyllide on exposure to  light.  If  ALA  is  given  to
plant  tissue  in  the   dark,   it   feeds   through   all   the   way   to
protochlorophyllide, but no further. This is because POR,  the  enzyme  that
converts protochlorophyllide to chlorophyllide, needs  light  to  carry  out
its reaction. POR is a very actively researched enzyme worldwide and  a  lot
is known about the chemistry and molecular  biology  of  its  operation  and
regulation. Much  less  is  known  about  how  POR  works  in  natural  leaf
development.

             ALA                                         Portoporphyrine



Protochlorophyllide



Chlophyllide


                Chlorophyll b                            Chlorophyll a


      Chlorophyll[3] is a green compound found in leaves and green stems of
plants. Initially, it was assumed that chlorophyll was a single compound
but in 1864 Stokes showed by spectroscopy that chlorophyll was a mixture.
If dried leaves are powdered and digested with ethanol, after concentration
of the solvent, 'crystalline' chlorophyll is obtained, but if ether or
aqueous acetone is used instead of ethanol, the product is 'amorphous'
chlorophyll.
      In 1912, Willstatter et al. (1) showed that chlorophyll was a mixture
of two compounds, chlorophyll-a and chlorophyll-b:
[pic]

Chlorophyll-a (C55H72MgN4O5, mol. wt.: 893.49). The methyl group marked
with an asterisk is replaced by an aldehyde in chlorophyll-b (C55H70MgN4O6,
mol. wt.: 906.51).


      The two components were separated by shaking a light petroleum
solution of chlorophyll with aqueous methanol: chlorophyll-a remains in the
light petroleum but chlorophyll-b is transferred into the aqueous methanol.
Cholorophyll-a is a bluish-black solid and cholorophyll-b is a dark green
solid, both giving a green solution in organic solutions. In natural
chlorophyll there is a ratio of 3 to 1 (of a to b) of the two components.
      The intense green colour of chlorophyll is due to its strong
absorbencies in the red and blue regions of the spectrum, shown in fig. 1.
(2) Because of these absorbencies the light it reflects and transmits
appears green.

[pic]

Fig. 1 - The uv/visible adsorption spectrum for chlorophyll.

      Due to the green colour of chlorophyll, it has many uses as dyes and
pigments. It is used in colouring soaps, oils, waxes and confectionary.
      Chlorophyll's most important use, however, is in nature, in
photosynthesis. It is capable of channelling the energy of sunlight into
chemical energy through the process of photosynthesis. In this process the
energy absorbed by chlorophyll transforms carbon dioxide and water into
carbohydrates and oxygen:

CO2 + H2O [pic](CH2O) + O2

Note: CH2O is the empirical formula of carbohydrates.

      The chemical energy stored by photosynthesis in carbohydrates drives
biochemical reactions in nearly all living organisms.
      In the photosynthetic reaction electrons are transferred from water to
carbon dioxide, that is carbon dioxide is reduced by water. Chlorophyll
assists this transfer as when chlorophyll absorbs light energy, an electron
in chlorophyll is excited from a lower energy state to a higher energy
state. In this higher energy state, this electron is more readily
transferred to another molecule. This starts a chain of electron-transfer
steps, which ends with an electron being transferred to carbon dioxide.
Meanwhile, the chlorophyll which gave up an electron can accept an electron
from another molecule. This is the end of a process which starts with the
removal of an electron from water. Thus, chlorophyll is at the centre of
the photosynthetic oxidation-reduction reaction between carbon dioxide and
water.

      Treatment of cholorophyll-a with acid removes the magnesium ion
replacing it with two hydrogen atoms giving an olive-brown solid,
phaeophytin-a. Hydrolysis of this (reverse of esterification) splits off
phytol and gives phaeophorbide-a. Similar compounds are obtained if
chlorophyll-b is used.

[pic]


      Chlorophyll can also be reacted with a base which yields a series of
phyllins, magnesium porphyrin compounds. Treatment of phyllins with acid
gives porphyrins.

[pic]


      Now knowing all these factors affecting the synthesis and  destruction
of chlorophyll I propose that the amount of chlorophyll in plant depends  on
light intensity in the following way: with the increase of  light  intensity
the amount of chlorophyll increases, but then it starts  decreasing  because
light intensity exceed the point when there is more  chlorophyll  destructed
than formed.


[pic]

                                 Variables.

Independent:
    . Light intensity, lux
Constant:
    . pH of soil
    . water supply, ml
    . temperature, to C
Dependent:
    . length, cm
    . amount of chlorophyll in gram of a plant, gram per gram



                                III. Method.

Apparatus:
            . seeds of Cicer arietinum
            . 28 plastic pots
            . water
            . scissors
            . ruler (20 cm ( 0.05 cm)
            . CaCO3
            . soil (adopted for home plants)
            . digital luxmeter (( 0.05 lux)
            . test tubes
            . H2SO4 (0.01 M solution)
            . Pipette (5 cm3 ( 0.05 cm3)
            . mortar and pestle
            . burette
            . ethanol (C2H5OH), 98%
            . beakers


   Firstly I  went  to  the  shop  and  bought  germinated  seeds  of  Cicer
arietinum. Then sorted seeds and chose the  strongest  ones.  After  that  I
prepared soil for them and put them in it.
   As the aim of this project is to investigate the dependence  of  mass  of
chlorophyll in plants during different light intensities it  was  needed  to
create those various conditions.  Pots  with  seeds  were  placed  into  the
following places: in the wardrobe with doors (light  intensity  is  o  lux),
under the sink (light intensity is 20,5  lux),  in  the  shell  of  bookcase
(light intensity is 27,5 lux), above the bookcase (light intensity  is  89,5
lux), above the extractor (light intensity is 142 lux), beyond the  curtains
(light intensity is 680 lux) and on the open sun (light  intensity  is  1220
lux). Light intensity was measured with the help  of  digital  luxmeter.  It
was measured four times  each  day:  morning,  midday,  afternoon,  evening.
During those four periods four measurements were done and the maximum  value
was taken into consideration and written  down.  Those  measurements  lasted
for three weeks of the experiment as the whole time of  the  experiment  was
three weeks. The luxmeters sensitive part was placed on the plants  (so  it
was just lying  on  them)  in  order  to  measure  light  intensity  flowing
directly on plant bodies, then  two  minutes  were  left  in  order  to  get
stabilized value of light intensity and the  same  procedure  was  repeated.
All those actions were done in order to get more accurate results  of  light
intensity.
   Growing plants were provided with the same amount of water (15 ml, once a
day in the morning) and they were situated  in  the  same  room  temperature
(20o C), pH of soil was definitely the same because all the plants were  put
in the same soil (special soil for room flowers).
   After three weeks past the length of plants was measured with the help of
ruler. Firstly the plants were not cut, so their length had to  be  measured
while they were in the pots. The ruler was placed into the  pot  and  plants
were carefully stretched on it. The action  was  repeated  three  times  and
only maximum value was taken into  consideration.  After  that  plants  were
cut. Then those already cut plants were put into the dark place and  quickly
dried.
   Titration.
   I have chosen three plants from each light intensity group  and  measured
their weight. . In order to obtain the pigments, three plants were cut  into
small pieces and placed in a  mortar.  Calcium  carbonate  was  then  added,
together with a little ethanol (2 cm3). The leaf was grinded using a  pestle
until no large pieces of leaf tissue were left, and  the  remaining  ethanol
was poured into the mortar (3 cm3). Then  1  ml  of  obtained  solution  was
placed into the test tube and this  1  ml  of  solution  was  then  titrated
against 0.01 M solution of sulfuric acid, through the use of a pipette.  The
titration was complete when the green solution turned  dark  olive-green[4].
This solution obtained from the first action was stored as  the  etalon  for
the  following  ones.  The  settled  olive-green  coloring  meant  that  all
chlorophyll had  reacted  with  H2SO4.  So  the  process  of  titration  was
repeated 7 times for all light intensity groups.
   The solution is titrated until the dark olive-green color because  it  is
known that when the reaction between chlorophyll and sulfuric acid  happens,
chlorophyll turns into phaeophetin which  has  grey  color  (see  table  1),
therefore  when  the  solution  is  olive-green,  than  the   reaction   has
succeeded. But while searching in the internet and books I  found  out  that
there are several opinions about the color of  phaeophytin    in  the  book
written by Viktorov it is ssaid to have grey  color,  but  in  the  internet
link    contact://www.ch.ic.ac.uk/local/projects/steer/chloro.php it  is  said  to
have brown olive-green  color.  Also  I  made  chromatography  in  order  to
investigate the color of phaeophytin and the result was  that  it  has  grey
color. It can be proposed that olive-green color is  obtained  because  grey
phaeophetyn is mixed with other plant pigments.
    So titration is one of the visual methods that can be used in  order  to
find the mass of chlorophyll in plants.
   All the measurements and even chromatography were done  three  times  and
the mean value was taken, for chromatography grey color was confirmed.



   Table 1. Plant pigments.

|Name of the pigment                 |Color of the pigment                |
|Chlorophylls ( a and b )            |Green                               |
|Carotene                            |Orange                              |
|Xanitophyll                         |Yellow                              |
|Phaeophytin-a                       |OLIVE BROUN or GREY                 |


                                IV. Results.

Table 2. Raw data.


|Number of    |Light intensity (lux)                                     |
|plant        |                                                          |
|0       |0,273       |0,041         |84,98       |41,89       |0,0000     |
|20,5    |0,579       |0,056         |90,33       |41,76       |0,0496     |
|27,5    |0,332       |0,033         |90,06       |36,33       |0,1462     |
|89,5    |0,181       |0,018         |90,06       |19,81       |0,1769     |
|142     |0,511       |0,047         |90,80       |41,33       |0,0697     |
|680     |0,338       |0,043         |87,28       |29,33       |0,1557     |
|1220    |0,301       |0,034         |88,70       |18,64       |0,1939     |

[pic]



Calculation of amount of chlorophyll in plants  basing  on  the  results  of
titration


H2 SO4  + C56 O5 N4 Mg => C56 O5 N4 H + MgSO4
Concentration of H2SO4 is 0,01 M
C  concentration
V  volume
n  quantity of substancy
m  mass
Mr  molar mass

For light intensity equal to 20,5 lux.
n = V (in dm3) ? C
2 ? 10-3 ? 0,01 = 2 ? 10-5
n = m / Mr => m = n ? Mr
m = 2 ? 10-5 ? 832 = 1,664 ? 10-2 grams
mass of plant                           mass of chlorophyll
1,68  grams                     -                       0,08335   grams   of
chlorophyll
1   gram                            -                        x   grams    of
chlorophyll
Hence there are 0,0496 grams of chlorophyll.
[pic]

Table 5. The correlation between mean length of plants and mean dry
biomass.



 | | | | | | | |


 | | | | | | | |



                                    [pic]

Table 6. The correlation between mean length and mass of chlorophyll per 1
g of plant.

Site  |Mean length, cm |Rank (R1) |Mass of chl. In 1 g |Rank (R2) |D (R1-
    R2) |D^2 | |1 |41,89 |1 |0,0000 |7 |-6 |36 | |2 |41,76 |2 |0,0496 |6 |-4
   |16 | |3 |36,33 |4 |0,1462 |4 |0 |0 | |4 |19,81 |6 |0,1769 |2 |4 |16 | |5
  |41,33 |3 |0,0697 |5 |-2 |4 | |6 |29,33 |5 |0,1557 |3 |2 |4 | |7 |18,64 |7
|0,1939 |1 |6 |36 | | | | | | | | | |
Rs = -1

                       | | | | | | | | | | | | | | | |


                                    [pic]

Table 7. The correlation between mean dry biomass and mass of chlorophyll
per 1 g of plant.

Site  |Mean dry biomass, g |Rank (R1) |Mass of chl. In 1 g |Rank (R2) |D
 (R1-R2) |D^2 | |1 |0,041 |4 |0,0000 |7 |-3 |9 | |2 |0,056 |1 |0,0496 |6 |-5
   |25 | |3 |0,033 |6 |0,1462 |4 |2 |4 | |4 |0,018 |7 |0,1769 |2 |5 |25 | |5
  |0,047 |2 |0,0697 |5 |-3 |9 | |6 |0,043 |3 |0,1557 |3 |0 |0 | |7 |0,034 |5
|0,1939 |1 |4 |16 | | | | | | | | | | | | | | | | | |Rs = -0,57 | | | | | |
| |


               | | | | | | | | | | | | | | | | | | | | | | | |



                                    [pic]

                               V. Discussion.
      Several tendencies can be clearly seen.
      For the first, with the increase of light  intensity  mean  length  of
plants is decreasing, but there are exceptions. For light intensity 142  lux
the value of mean length is approximately equal to the values of length  for
light intensities 0 lux and 20,5 lux. If exclude this data it is  also  seen
that for light intensity equal to 680  lux  mean  length  is  also  slightly
falling out from the main tendency  decreasing from 19.81 cm.
      The second tendency is increase of mass of chlorophyll per 1  gram  of
plant biomass with the increase of light intensity. But the values  of  mass
of chlorophyll of those plants under light intensities 142 lux and  680  lux
are falling out from the main tendency. The first and the  second  ones  are
too small  approximately equal to  the  value  corresponding  to  20.5  lux
light intensity and to 89.5 lux respectively. This may  happen  because  not
all the seeds of Cicer arietnum were of the  same  quality,  because  it  is
impossible to guarantee that more than 250 seeds in one box  have  the  same
high quality. At the mean time it was expected that starting from the  light
intensity more than 680  lux  the  amount  of  chlorophyll  in  plants  will
decrease, because the value of destructed chlorophyll with  be  bigger  than
the value of newly formatted. But the experiments showed that the amount  of
chlorophyll was constantly increasing even when the  light  intensity  level
exceeded the point 1220 lux.  This  could  happen  because  light  intensity
equal to 1220 lux is  not  so  extremely  high  that  the  amount  of  total
chlorophyll in plants will start decreasing.
      Also it is clearly seen that there are no correlations  between  light
intensity and values of wet and dry biomass.
      Basing on these  arguments  the  sudden  decrease  of  the  amount  of
chlorophyll in plants placed on light intensity equal to 142 lux was  likely
to be insignificant and could not be considered as a trend.
    But it is impossible to forget such important factor as plant  hormones
that affect the growth and development of plants. There are  five  generally
accepted types of hormones that  influence  plant  growth  and  development.
They are: auxin, cytokinin, gibberellins, abscic acid, and ethylene.  It  is
not one hormone that directly influences by sheer quantity. The balance  and
ratios of hormones present is what helps to influence plant  reactions.  The
hormonal balance possibly regulates enzymatic  reactions  in  the  plant  by
amplifying them.



VI. Conclusion.
      Due to results of my investigation  it  is  seen  that  my  hypothesis
didnt confirm fully (for example, comparing the diagram 1 and  diagram  7),
because I proposed that when light intensities will be very  high,  mass  of
chlorophyll in plant will start decreasing and due  to  my  observations  it
didnt happen. I should say that the only reason I can  suggest  is  that  I
havent  investigated  such  extremely  high  light  intensities,  so   that
chlorophyll start destructing. But if we will  not  pay  attention  to  that
fact the other part of my hypothesis was confirmed and mass  of  chlorophyll
in plants increased with the increase  of  light  intensity.  Furthermore  I
didnt estimate amount of  plant  hormones  and  so  didnt  estimate  their
influence on results.

      Questions for further investigation:
          1. Investigating very high light intensities.
          2. Implementation of colorimetric analysis.
          3. Paying attention to estimation of plant hormones level.

      Those questions should be further investigated in order to get clearer
picture and more accurate  results  of  the  dependence  of  the  amount  of
chlorophyll in plants on the light intensity,  knowing  the  fact  that  the
amount of chlorophyll has a tendency to decrease  at  extremely  high  light
intensities. So this statement needs an experimental confirmation and as  in
this investigation  conditions  with  extremely  light  intensity  were  not
created in further investigations they have to be created.
    Implementation of colorimetric analysis is also very  important  thing,
because it gives much more accurate results  comparing  with  the  titration
method. The colorimetric method suggests that as different  pigments  absorb
different parts of light spectrum differently, the absorbance of a  pigments
mixture is a sum of individual absorption spectra.  Therefore  the  quantity
of each individual pigment in a mixture can be calculated  using  absorbance
of the certain colors and molecular coefficients of each pigment.  This  was
proposed by D. A. Sims,  and  J.  A.  Gamon  (California  State  University,
USA)[5] with the reference on Lichtenthaler (1987).

VII. Evaluation.
      There are several results in my work, that are falling  out  from  the
main tendencies. It may seem that such results may occur  due  to  different
percentage  of  water  in  plants,  but  when  I  was  calculating  mass  of
chlorophyll in 1 gram of plant I was using only values of mean  dry  biomass
so it couldnt affect my results. (see table 3)
      At the same time such differences  in  the  percentage  of  water  are
easily explained. The rate of evaporation of water from plants,  which  were
put under 1220 lux light intensity was much higher than of those  put  under
20.5 lux light intensity, therefore percentage of  water  in  the  soil  may
vary, though I provided all the plants with the same volume of water at  the
same periods of time.
      One more reason that could be proposed is the  reason  connected  with
the pH of water with which flowers were provided. It was  not  measured  but
the thing that could have happened is that it had somehow changed the pH  of
soil in which  seeds  were  placed  and  therefore  changed  the  amount  of
synthesized chlorophyll.
      Titration is not a perfect way  of  obtaining  results.  This  happens
because the method is based on visual abilities of a  person    he  has  to
decide whether the color he obtained is dark  olive-green  or  not  so  dark
olive-green. Such a situation concerns lots of  mistakes  due  to  different
optical abilities  of  each  person,  even  some  humans  are  not  able  to
distinguish those colors, because of the disease called Daltonism.
      Even those who do not suffer from this disease can also make  mistakes
in such experiment. It is known that  people  who  suffer  from  Myopia  can
hardly see objects that are far from them,  but  dont  have  problems  with
objects that are near, but it is also important to take  into  consideration
the fact that their ability to distinguish colors is  also  lower  comparing
with humans with normal eyesight.
      There also exist the so called human factor, which  also  affects  the
investigation. Man cant be absolutely objective, because  sometimes  it  is
too hard for a person to falsify his own theory or hypothesis,  so  one  can
ignore results that are not suitable for  his  statements  and  select  only
those that are suitable, which will also affect  the  investigation  not  in
good way.
       So as humans eye is not a perfect  instrument  and  humans  are  not
perfectly objective there should  be  other  methods  of  investigating  the
amount of chlorophyll in plant.
      Moreover titration method doesnt distinguish  between  chlorophylls-a
and chlorophyll-b, phaeophytin-a and phaeophytin-b, as their colors  differ,
this giving not very accurate results. Also due to this limiting  factor  it
is impossible to know whether the whole amount of chlorophyll  reacted  with
the sulfuric  acid  and  again  it  adds  an  uncertainty  to  the  results.
Furthermore the saturation of color depends on the extent  of  dilution  and
it is nearly impossible to say if the solution was  diluted  till  the  same
color or not, because it is very difficult to distinguish between  different
shades of olive green color.



BIBLIOGRAPHY

   1) Allott, Biology for IB diploma (standard and higher level), Oxford
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   6)    contact://vcsars.calstatela.edu/esa_posters/ds/dan_esa99.phpl 05/05/2004
   7)    contact://www.agsci.ubc.ca/courses/fnh/410/colour/3_21.php, 16/03/2004
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   9)    contact://www.charlies-web.com/specialtopics/anthocyanin.phpl. 17/04/2004
  10)    contact://www.ch.ic.ac.uk/local/projects/steer/chloro.php, 11/04/2004
  11)    contact://www.bonsai.ru/dendro/physiology5.phpl 02/04/2004
  12)    contact://www.iger.bbsrc.ac.uk/Publications/Innovations/in97/Ch2.pdf,
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-----------------------
[1]    contact://www.bonsai.ru/dendro/physiology5.phpl 02/04/2004

[2] www.iger.bbsrc.ac.uk/igdev/iger_innovations/ 06/05/2004
[3]    contact://www.ch.ic.ac.uk/local/projects/steer/chloro.php 11/04/2004
[4] 8:B>@>2 . ., @0:B8:C< ?> D878>;>388  . .,  
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[5]    contact://vcsars.calstatela.edu/esa_posters/ds/dan_esa99.phpl 05/05/2004

-----------------------
Chlorophyll, gram per gram of plant.

Light intensity, lux

Diagram 1. The predicted change of amount of chlorophyll in leaves of
depending on light intensity

0,57<0,79, therefore there is no significant correlation between mean
length of plants and mean dry biomass.

POR

max

plateau

There is negative correlation between mean length of plants and mass of
chlorophyll per 1 g of plant

0,57<0,79, therefore there is no significant correlation between mean dry
biomass and mass of chlorophyll per D[pic]D[pic]1 g of plant