Реферат: The effect of light intensity on the amount of chlorophyll in “Cicer arietinum”
“The effect of light intensity on the amount ofchlorophyll in “Cicer arietinum”
Wordcount: 4 413 wordsContent
Evaluation of themethod ………………………………………………………..…… 15
Plants, growing on the shaded area has less concentratedgreen color and are much longer and thinner than plants growing on the sunareas as they are dark green, short and thick. Research question was: “How doesthe amount of chlorophyll-a and chlorophyll-b, gram per gram ofplant, depends on the light intensity in which plants are placed?”
Hypothesis suggeststhat there are several inner and outer factors that affect the amount ofchlorophylls a and b in plants and that with the increase oflight intensity the amount of chlorophyll will also increase until lightintensity exceeds the value when the amount of destructed chlorophylls isgreater than formatted thus decreasing the total amount of chlorophylls in aplant.
The seeds of Cicerarietinum were divided into seven groups and placed into various placeswith different values of light intensities. Light intensities were measuredwith digital colorimeter. After three weeks length was measured. Then plantswere cut and quickly dried. Their biomass was also measured. Three plants fromeach group were grinded and the ethanol extract of pigments was prepared. Theamount of chlorophylls was measured using method of titration and differentformulas.
The investigationshowed that plants growing on the lowest light intensity equal 0 lux containedno chlorophyll and had the longest length. The amount of chlorophyll quicklyincreased and length decreased with the increase of light intensity from 0 luxto 1200 lux. The amount of chlorophyll in plants unpredictably decreased duringlight intensity equal to 142 lux and than continued increasing and didn’t startdecreasing reaching very high value (1200 lux).
The sudden decreasehappened due to mighty existence of some inner genetical damages of seeds whichprevented them from normal chlorophyll synthesis and predicted decrease didn’tdecrease because extremely high light intensity was not exceeded.
Word count: 300 words
Thistheme seemed to be attractive for me because I could see that results of myinvestigation could find application in real life.
Whilewalking in the forest in summer I saw lots of plants of different shades of greencolor: some of them were dark green, some were light green and some evenvery-very light green with yellow shades, hence I became very interested inthis situation and wanted to know why it happens to be so. I also saw thatthose plants that were growing on sunny parts of forest, where trees were notvery high, had dark green color and those, that were growing in shady parts ofthe same forest had very light green color. They also had difference in theirlength and thickness – those, that were growing on light were very short, butthick and strong, and those, growing in shady regions were very thin andfragile.
HenceI became very interested in finding scientifical description of myobservations.
Theaim of my project is to find out how does the changes in light intensity affectbalance of chlorophyll in Cicer arietinum.
There are several factors that affect the development of chlorophyllin plants.
Inner factors. The most important one is– genetical potential of a plant, because sometimes happen mutations thatfollow in inability of chlorophyll formation. But most of the times it happensthat the process of chlorophyll synthesis is broken only partly, revealing inabsence of chlorophyll only in several parts of the plant or in general lowrate of chlorophyll. Therefore plants obtain yellowish color. Lots of genesparticipate in the process of chlorophyll synthesis, therefore differentanomalies are widely represented. Development of chloroplasts depends onnuclear and plastid DNA and also on cytoplasmatic and chloroplastic ribosomes.
Full provision of carbohydrates seem to be essential for chlorophyllformation, and those plants that suffer from deficit of soluble carbohydratesmay not become green even if all other conditions are perfect. Such leaves,placed into sugar solution normally start to form chlorophyll. Very often ithappens that different viruses prevent chlorophyll formation, causing yellowcolor of leaves.
Outsidefactors.The most important outside factors, affecting the formation of chlorophyll are:light intensity, temperature, pH of soil, provision of minerals, water andoxygen. Synthesis of chlorophyll is very sensitive to all the factors,disturbing metabolic processes in plants.
Light. Light is veryimportant for the chlorophyll formation, though some plants are able to producechlorophyll in absolute darkness. Relatively low light intensity is rathereffective for initialization and speeding of chlorophyll development. Greenplants grown in darkness have yellow color and contain protochlorophyll –predecessor of chlorophyll а, which needs lite torestore until chlorophyll а. Very high lightintensity causes the destruction of chlorophyll. Hence chlorophyll issynthesized and destructed both at the same time. In the condition of very highlight intensity balance is set during lower chlorophyll concentration, than incondition of low light intensity.
Temperature. Chlorophyll synthesisseems to happen during rather broad temperature intervals. Lots of plants of умеренной зоныsynthesize chlorophyll from very low temperatures till very high temperaturesin the mid of the summer. Many pine trees loose some chlorophyll during wintersand therefore loose some of their green color. It may happen because thedestruction of chlorophyll exceeds its formation during very low temperatures.
Provisionwith minerals.One of the most common reason for shortage of chlorophyll is absence of someimportant chemical elements. Shortage of nitrogen is the most common reason forlack of chlorophyll in old leaves. Another one is shortage of ferrum, mostly inyoung leaves and plants. And ferrum is important element for chlorophyllsynthesis. And magnesium is a component of chlorophyll therefore its shortagecauses lack of production of chlorophyll.
Water. Relatively low water stresslowers speed of chlorophyll synthesis and high dehydration of plants tissuesnot only disturbs synthesis of chlorophyll, but even causes destruction ofalready existing molecules.
Oxygen. With the absence of oxygen plants do not produce chlorophyll even onhigh light intensity. This shows that aerobic respiration is essential forchlorophyll synthesis.
/> Chlorophyll.The synthesis ofchlorophyll is induced by light. With light, a gene can be transcripted andtranslated in a protein.
The plants arenaturally blocked in the conversion of protochlorophyllide to chlorophyllide.In normal plants these results in accumulation of a small amount ofprotochlorophyllide which is attached to holochrome protein. In vivo at leasttwo types of protochlorophyllide holochrome are present. One, absorbingmaximally at approximately 650 nm, is immediately convertible to chlorophyllideon exposure to light. If ALA is given to plant tissue in the dark, it feedsthrough all the way to protochlorophyllide, but nofurther. This is because POR, the enzyme that converts protochlorophyllideto chlorophyllide, needs light to carry outits reaction. POR is a very actively researched enzyme worldwide and a lot isknown about the chemistry and molecular biology of its operation andregulation. Much less is known about how POR works in natural leaf development.
/> ALA Portoporphyrine
/>/> Chlorophyll b Chlorophylla
Chlorophyll is a green compoundfound in leaves and green stems of plants. Initially, it was assumed thatchlorophyll was a single compound but in 1864 Stokes showed by spectroscopythat chlorophyll was a mixture. If dried leaves are powdered and digested withethanol, after concentration of the solvent, 'crystalline' chlorophyll isobtained, but if ether or aqueous acetone is used instead of ethanol, theproduct is 'amorphous' chlorophyll.
In 1912,Willstatter et al. (1) showed that chlorophyll was a mixture of twocompounds, chlorophyll-a and chlorophyll-b:
Chlorophyll-a (C55H72MgN4O5,mol. wt.: 893.49). The methyl group marked with an asterisk is replaced by analdehyde in chlorophyll-b (C55H70MgN4O6,mol. wt.: 906.51).
The twocomponents were separated by shaking a light petroleum solution of chlorophyllwith aqueous methanol: chlorophyll-a remains in the light petroleum butchlorophyll-b is transferred into the aqueous methanol. Cholorophyll-ais a bluish-black solid and cholorophyll-b is a dark green solid, bothgiving a green solution in organic solutions. In natural chlorophyll there is aratio of 3 to 1 (of a to b) of the two components.
The intensegreen colour of chlorophyll is due to its strong absorbencies in the red andblue regions of the spectrum, shown in fig. 1. (2) Because of these absorbenciesthe light it reflects and transmits appears green.
Fig. 1 — The uv/visible adsorption spectrum for chlorophyll.
Due to the greencolour of chlorophyll, it has many uses as dyes and pigments. It is used incolouring soaps, oils, waxes and confectionary.
Chlorophyll'smost important use, however, is in nature, in photosynthesis. It is capable ofchannelling the energy of sunlight into chemical energy through the process ofphotosynthesis. In this process the energy absorbed by chlorophyll transformscarbon dioxide and water into carbohydrates and oxygen:
CO2 + H2O />(CH2O) + O2
Note: CH2O is theempirical formula of carbohydrates.
The chemicalenergy stored by photosynthesis in carbohydrates drives biochemical reactionsin nearly all living organisms.
In thephotosynthetic reaction electrons are transferred from water to carbon dioxide,that is carbon dioxide is reduced by water. Chlorophyll assists this transferas when chlorophyll absorbs light energy, an electron in chlorophyll is excitedfrom a lower energy state to a higher energy state. In this higher energystate, this electron is more readily transferred to another molecule. Thisstarts a chain of electron-transfer steps, which ends with an electron beingtransferred to carbon dioxide. Meanwhile, the chlorophyll which gave up anelectron can accept an electron from another molecule. This is the end of aprocess which starts with the removal of an electron from water. Thus,chlorophyll is at the centre of the photosynthetic oxidation-reduction reactionbetween carbon dioxide and water.
Treatment ofcholorophyll-a with acid removes the magnesium ion replacing it with twohydrogen atoms giving an olive-brown solid, phaeophytin-a. Hydrolysis ofthis (reverse of esterification) splits off phytol and gives phaeophorbide-a.Similar compounds are obtained if chlorophyll-b is used.
Chlorophyll canalso be reacted with a base which yields a series of phyllins, magnesiumporphyrin compounds. Treatment of phyllins with acid gives porphyrins.
Now knowing all these factors affecting the synthesis anddestruction of chlorophyll I propose that the amount of chlorophyll in plantdepends on light intensity in the following way: with the increase of lightintensity the amount of chlorophyll increases, but then it starts decreasingbecause light intensity exceed the point when there is more chlorophylldestructed than formed.
Diagram 1. The predicted change of amount of chlorophyll in leaves of depending on light intensity
Light intensity, lux
Chlorophyll, gram per gram of plant./>/>
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
· seeds of Cicer arietinum
· 28 plastic pots
· ruler (20 cm ± 0.05 cm)
· 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
· ethanol (C2H5OH),98%
Firstly I went to the shop and boughtgerminated seeds of Cicer arietinum. Then sorted seeds and chose thestrongest ones. After that I prepared soil for them and put them in it.
As the aim of this project is toinvestigate the dependence of mass of chlorophyll in plants during differentlight intensities it was needed to create those various conditions. Pots withseeds were placed into the following places: in the wardrobe with doors (lightintensity is o lux), under the sink (light intensity is 20,5 lux), in the shellof bookcase (light intensity is 27,5 lux), above the bookcase (light intensityis 89,5 lux), above the extractor (light intensity is 142 lux), beyond thecurtains (light intensity is 680 lux) and on the open sun (light intensity is1220 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 themaximum value was taken into consideration and written down. Those measurementslasted for three weeks of the experiment as the whole time of the experimentwas three weeks. The luxmeter’s sensitive part was placed on the plants (so itwas just lying on them) in order to measure light intensity flowing directly onplant bodies, then two minutes were left in order to get stabilized value oflight intensity and the same procedure was repeated. All those actions weredone in order to get more accurate results of light intensity.
Growing plants were provided with the sameamount of water (15 ml, once a day in the morning) and they were situated inthe same room temperature (20o C), pH of soil was definitely thesame because all the plants were put in the same soil (special soil for roomflowers).
After three weeks past the length ofplants was measured with the help of ruler. Firstly the plants were not cut, sotheir length had to be measured while they were in the pots. The ruler wasplaced into the pot and plants were carefully stretched on it. The action was repeatedthree times and only maximum value was taken into consideration. After thatplants were cut. Then those already cut plants were put into the dark place andquickly dried.
I have chosen three plants from each lightintensity group and measured their weight… In order to obtain the pigments,three plants were cut into small pieces and placed in a mortar. Calciumcarbonate was then added, together with a little ethanol (2 cm3).The leaf was grinded using a pestle until no large pieces of leaf tissue wereleft, 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 ofsolution was then titrated against 0.01 M solution of sulfuric acid, throughthe use of a pipette. The titration was complete when the green solution turneddark olive-green.This solution obtained from the first action was stored as the etalon for thefollowing ones. The settled olive-green coloring meant that all chlorophyll hadreacted with H2SO4. So the process of titration wasrepeated 7 times for all light intensity groups.
The solution is titrated until the darkolive-green color because it is known that when the reaction betweenchlorophyll and sulfuric acid happens, chlorophyll turns into phaeophetin whichhas grey color (see table 1), therefore when the solution is olive-green, thanthe reaction has succeeded. But while searching in the internet and books Ifound out that there are several opinions about the color of phaeophytin – inthe book written by Viktorov it is ssaid to have grey color, but in theinternet link www.ch.ic.ac.uk/local/projects/steer/chloro.htm it is said to have brown olive-green color. Also I made chromatography in orderto investigate the color of phaeophytin and the result was that it has greycolor. It can be proposed that olive-green color is obtained because greyphaeophetyn is mixed with other plant pigments.
So titration is one of the visual methods that can be used in order tofind the mass of chlorophyll in plants.
All the measurements and evenchromatography were done three times and the mean value was taken, forchromatography grey color was confirmed.
Table 1. Plant pigments.
Name of the pigment
Color of the pigment
Chlorophylls ( a and b )
OLIVE BROUN or GREY
Table 2. Raw data.
Number of plant
Light intensity (lux)0,0 20,5 27,5 89,5 142,0 680,0 1220,0 1 23 35 20 1 30 2 15 2 30 36 33 4 31 20 16 3 38 37 35 8 34 21 16 4 39 37 36 9 35 21 16 5 44 38 37 9 38 21 17 6 46 39 40 12 38 22 17 7 50 39 40 12 38 22 19 8 52 40 43 13 39 23 20 9 55 40 43 15 39 25 21 10 40 18 40 27 22 11 42 20 41 29 26 12 42 22 41 30 13 42 22 41 31 14 42 24 42 33 15 43 25 42 34 16 43 25 43 34 17 44 25 43 35 18 44 25 43 35 19 45 26 45 37 20 45 26 45 38 21 45 26 46 38 22 45 26 46 41 23 46 27 48 41 24 46 29 48 44 25 49 32 49 26 34 49
Mean41,888889 41,76 36,33333 19,80769 41,30769 29,33333 18,63636
Median44 42 37 23 41,5 30,5 17
St.deviation10,50529 2,928 4,740741 7,467456 4 7,472222 2,694215
Table 3. Frequency of lengths of3-weeks-old plants depending on light intensity.
Light intensity, lux
Plant length, cm (class) 0,0 20,5 27,5 89,5 142,0 680,0 1220,0 0.0-10.0 5 1 10.1-20.0 1 6 1 8 20.1-30.0 2 13 1 10 3 30.1-40.0 2 9 6 2 9 9 40.1-50.0 3 15 2 16 3 50.1-60.0 2 Total 9 24 9 26 26 24 11
Table 3 (alternative) Frequency of lengthof 3-weeks-old plants depending on light intensity.Light intensity, lux Plant length (Class) 0,0 20,5 27,5 89,5 142,0 680,0 1220,0 0.0-10.0 19,23% 4,17% 10.1-20.0 11,10% 23,08% 4,17% 72,72% 20.1-30.0 50% 3,85% 41,62% 27,28% 30.1-40.0 37,50% 66,60% 7,69% 34,62% 37,52% 40.1-50.0 62,50% 22,30% 61,53% 12,52% 50.1-60.0 100% Total 1 1 1 1 1 1 1
Calculationof the mean length of plants.
For lightintensity equal to 20,50 lux:
The sum oflengths of all plants in this group is 45cm + 37cm + 39cm + 49cm + 46cm + 44cm+ 45cm + 44cm + 42cm + 37cm + 40cm + 40cm + 39cm + 43cm + 42cm + 42cm + 36cm +45cm + 38cm + 45cm + 46cm + 40cm + 35cm + 42cm + 43cm = 1044cm
Hence meanlength is 1044cm: 25 plants = 41,76cm
Light intensity, lux
Mean wet biomass, g
Mean dry biomass, g
% of water
Mean length, cm
Mass of chl. In 1 g0,273 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
/>Calculation of amount of chlorophyll in plants basing onthe results of titration
H2 SO4 + C56 O5 N4 Mg => C56 O5N4 H + MgSO4
Concentration ofH2SO4 is 0,01 M
V – volume
n – quantity ofsubstancy
m – mass
Mr – molar mass
For lightintensity 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 ofplant mass of chlorophyll
1,68grams - 0,08335 grams of chlorophyll
1 gram - x grams of chlorophyll
Hence there are0,0496 grams of chlorophyll.
Table 5. The correlation between meanlength of plants and mean dry biomass.Site Mean length, cm Rank (R1) Mean dry biomass, g Rank (R2) D (R1-R2)
D21 41,89 1 0,041 4 -3 9 2 41,76 2 0,056 1 1 1 3 36,33 4 0,033 6 -2 4 4 19,81 6 0,018 7 -1 1 5 41,33 3 0,047 2 1 1 6 29,33 5 0,043 3 2 4 7 18,64 7 0,034 5 2 4 Rs = 0,57 critical value = 0,79 <p/>
Table 6. The correlation between meanlength 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 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 <p/>
Table 7. The correlation between mean drybiomass 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 7 0,034 5 0,1939 1 4 16 Rs = -0,57 <p/>
Several tendencies can be clearly seen.
For the first, with the increase of light intensity mean length ofplants is decreasing, but there are exceptions. For light intensity 142 lux thevalue of mean length is approximately equal to the values of length for lightintensities 0 lux and 20,5 lux. If exclude this data it is also seen that forlight intensity equal to 680 lux mean length is also slightly falling out fromthe main tendency – decreasing from 19.81 cm.
The second tendency is increase of mass of chlorophyll per 1 gram ofplant biomass with the increase of light intensity. But the values of mass ofchlorophyll of those plants under light intensities 142 lux and 680 lux arefalling 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 intensityand to 89.5 lux respectively. This may happen because not all the seeds of Cicerarietnum were of the same quality, because it is impossible to guaranteethat more than 250 seeds in one box have the same high quality. At the meantime it was expected that starting from the light intensity more than 680 luxthe amount of chlorophyll in plants will decrease, because the value ofdestructed chlorophyll with be bigger than the value of newly formatted. Butthe experiments showed that the amount of chlorophyll was constantly increasingeven when the light intensity level exceeded the point 1220 lux. This couldhappen because light intensity equal to 1220 lux is not so extremely high thatthe amount of total chlorophyll in plants will start decreasing.
Also it is clearly seen that there are no correlations between lightintensity and values of wet and dry biomass.
Basingon these arguments the sudden decrease of the amount of chlorophyll in plantsplaced on light intensity equal to 142 lux was likely to be insignificant andcould not be considered as a trend.
But it isimpossible to forget such important factor as plant hormones that affect thegrowth and development of plants. There are five generally accepted types ofhormones that influence plant growth and development. They are: auxin,cytokinin, gibberellins, abscic acid, and ethylene. It is not one hormone thatdirectly influences by sheer quantity. The balance and ratios of hormonespresent is what helps to influence plant reactions. The hormonal balancepossibly regulates enzymatic reactions in the plant by amplifying them.
Dueto results of my investigation it is seen that my hypothesis didn’t confirmfully (for example, comparing the diagram 1 and diagram 7), because I proposedthat when light intensities will be very high, mass of chlorophyll in plantwill start decreasing and due to my observations it didn’t happen. I should saythat the only reason I can suggest is that I haven’t investigated suchextremely high light intensities, so that chlorophyll start destructing. But ifwe will not pay attention to that fact the other part of my hypothesis wasconfirmed and mass of chlorophyll in plants increased with the increase oflight intensity. Furthermore I didn’t estimate amount of plant hormones and so didn’testimate 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 hormoneslevel.
Those questions should be further investigated in order to getclearer picture and more accurate results of the dependence of the amount ofchlorophyll in plants on the light intensity, knowing the fact that the amountof chlorophyll has a tendency to decrease at extremely high light intensities.So this statement needs an experimental confirmation and as in thisinvestigation conditions with extremely light intensity were not created infurther investigations they have to be created.
Implementation of colorimetric analysis isalso very important thing, because it gives much more accurate resultscomparing with the titration method. The colorimetricmethod suggests that as different pigments absorb different parts of lightspectrum differently, the absorbance of a pigments mixture is a sum ofindividual absorption spectra. Therefore the quantity of each individualpigment in a mixture can be calculated using absorbance of the certain colors and molecularcoefficients of each pigment. This was proposed by D.A. Sims, and J. A. Gamon (California State University, USA) with the reference on Lichtenthaler (1987).
There are several results in my work, that are falling out from themain tendencies. It may seem that such results may occur due to differentpercentage of water in plants, but when I was calculating mass of chlorophyllin 1 gram of plant I was using only values of mean dry biomass so it couldn’taffect my results. (see table 3)
At the same time such differences in the percentage of water areeasily explained. The rate of evaporation of water from plants, which were putunder 1220 lux light intensity was much higher than of those put under 20.5 luxlight intensity, therefore percentage of water in the soil may vary, though Iprovided all the plants with the same volume of water at the same periods oftime.
One more reason that could be proposed is the reason connected withthe pH of water with which flowers were provided. It was not measured but thething that could have happened is that it had somehow changed the pH of soil inwhich seeds were placed and therefore changed the amount of synthesizedchlorophyll.
Titration is not a perfect way of obtaining results. This happensbecause the method is based on visual abilities of a person – he has to decidewhether 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 abilitiesof each person, even some humans are not able to distinguish those colors,because of the disease called Daltonism.
Eventhose who do not suffer from this disease can also make mistakes in suchexperiment. It is known that people who suffer from Myopia can hardly seeobjects that are far from them, but don’t have problems with objects that arenear, but it is also important to take into consideration the fact that theirability to distinguish colors is also lower comparing with humans with normaleyesight.
There also exist the so called human factor, which also affects theinvestigation. Man can’t be absolutely objective, because sometimes it is toohard for a person to falsify his own theory or hypothesis, so one can ignoreresults that are not suitable for his statements and select only those that aresuitable, which will also affect the investigation not in good way.
So as human’s eye is not a perfect instrument and humans are notperfectly objective there should be other methods of investigating the amountof chlorophyll in plant.
Moreovertitration method doesn’t distinguish between chlorophylls-a andchlorophyll-b, phaeophytin-a and phaeophytin-b, as theircolors differ, this giving not very accurate results. Also due to this limitingfactor it is impossible to know whether the whole amount of chlorophyll reactedwith 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 isnearly impossible to say if the solution was diluted till the same color ornot, because it is very difficult to distinguish between different shades of olivegreen color.
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