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. 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 luxmeter’s 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 http://www.ch.ic.ac.uk/local/projects/steer/chloro.htm 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.

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[pic]

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

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