Pema Deki, Pema Lhaden, Pema Tshomo, Pema Wangdi and Passang Wangdi
I. THEORY
Chlorophyll
Chlorophylls are green pigments present in a wide variety of plants, fungi, and cyanobacteria. Chlorophyll can be present in nearly any green component of a plant, such as the leaves and stems, as well as in the chloroplast, which is the only organelle that holds the majority of it. It is located in the center of plant leaves in the mesophyll base. It transforms solar energy into chemical energy, which is then used to build vital carbohydrate molecules (glucose) that serve as the plant’s primary food supply. Chlorophyll a and b are found abundant in all autotrophic plants. The molecular formula of chlorophyll is C55H72O5N4Mg as shown in Figure 1 (Pareek et al., 2018).
Figure 1: The showing the structure of chlorophyll with hydrophobic hydrocarbon chain (tail) and hydrophilic porphyrin (head) (Pareek et al., 2017).
Chlorophyll a is essential for most photosynthetic organisms to release chemical energy and used in oxygenic photosynthesis. It absorbs most of the energy from wavelengths of violet-blue and orange-red light and chlorophyll b absorbs light same to chlorophyll a. In vitro, the characteristic absorption peak of chlorophyll b was observed at 453 nm and 625 nm, and in vivo, at 480 nm and 650 nm as shown in Figure 2 (Asimovic et al., 2016).
Spectrophotometer
The simplest instrument for determining the concentration of chlorophyll in extracts of various plant specimens is a spectrophotometer. The instrument used is spectrophotometer and it measure how much a chemical substance absorbs light by measuring the light intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over certain range of wavelength (Shim, 2021).
The procedure to quantify spectrophotometric chlorophyll pigment a and b is based on absorption of light by chlorophyll extracts prepared in aqueous acetone 80% (Olena et al., 2019). The aqueous acetone breaks down the chlorophyll’s lipid bond from the thylakoid structure which suspends the chlorophyll pigment highly soluble in organic solvent (Johan et al., 2014).
Figure 2: The absorbance curve of chlorophyll a and b at different regions of light (Pareek et al., 2017).
Spectrophotometer
The simplest instrument for determining the concentration of chlorophyll in extracts of various plant specimens is a spectrophotometer. The instrument used is spectrophotometer and it measure how much a chemical substance absorbs light by measuring the light intensity of light as a beam of light passes through sample solution. The basic principle is that each compound absorbs or transmits light over certain range of wavelength (Shim, 2021). The procedure to quantify spectrophotometric chlorophyll pigment a and b is based on absorption of light by chlorophyll extracts prepared in aqueous acetone 80% (Olena et al., 2019). The aqueous acetone breaks down the chlorophyll’s lipid bond from the thylakoid structure which suspends the chlorophyll pigment highly soluble in organic solvent (Johan et al., 2014).
Figure 3: Material used in the experiment: A: Green leafy herbs. B: Spectrophotometer. C: Mortar and pestle.
| Apparatus used | Specimen used | Solution taken | |
| 1. Weighing machine | 8. Test-tubes (5 numbers) | 1. Opuntia sp | 1. 80% Acetone |
| 2. Measuring Cylinder (25ml) | 9. Petri plate | 2. Brassica juncea | 2. Distilled water |
| 3. Mortar and pestle | 10. Blade | 3. Fagopyrum dibotrys | |
| 4. Pestle | 11. Cuvette (2) | 4. Tropaeolum majus | |
| 5. Filter paper (4 numbers) | 12. Tissue paper | 5. Rumex nepalensis | |
| 6. Funnel (4 numbers) | 13. Dropper | ||
| 7. Conical flask (2) | 14. Spectrophotometer 106 |
PROCEDURE
- All the materials were collected.
- The spectrophotometer was switched on before 20 minutes.
- A 100ml of acetone of 80% concentration was prepared by adding 20ml distilled water in a conical flask and then added 80ml of acetone using a measuring cylinder.
- The midrib of Brassica juncea, Tropaeolum majus, Rumex nepalensis, Fagopyrum dibotrys and Opuntia sp. was removed using a blade.
- 1g of leaf of each specimen was measured using a weighing machine (Figure 4).
- 5ml of 80% acetone solution was added to the pestle along with the 1g of leaves of each specimens and it was grinded using a mortar. It was grinded thoroughly until the last bit of leaves were visible (Figure 5).
- Then added the 5ml of acetone and mixed again thoroughly.
- The mixture was filtered using a filter paper and funnel (Figure 5).
- The residue left on the filter paper was again grinded with addition of 5ml 80% acetone and filtered to obtain the enough filtered.
- The cuvette was cleaned and rinsed using a tissue paper.
- The filtrate was poured in the cuvette for the reading to be taken (Figure 5).
- The reading of absorbance was measured at two different wavelengths (663 and 645nm) using spectrophotometer 106.
- The amount of chlorophyll a, b and total (a + b) are determined using the formula given by Arnon (1949) as follows:
III. Result
Table 1: Amount of chlorophyll content
| Herbaceous plants | Brassica juncea | Fagopyrum dibotrys | Opuntia sp. | Rumex nepalensis | Tropaeolum majus |
| Chlorophyll a | 19.35 | 0.0037 | 3.75 | 3.13 | 10.52 |
| Chlorophyll b | 16.88 | 0.707 | 5.13 | 2.73 | 0.949334 |
| Total Chlorophyll (a + b) | 15.171 | 0.6326 | 4.58 | 2.45 | 8.98 |
Graph 1: The amount of chlorophyll a, b and total amount (a + b) in different herbaceous plants.
Table 2: Measurement of absorbance of each species.
| Wavelength (nm) | Brassica juncea | Fagopyrum dibotrys | Opuntia Sp. | Rumex nepalensis | Tropaeolum majus |
| 645 | 0.608 | 0.041 | 0.233 | 0.124 | 0.44 |
| 650 | 0.937 | 0.031 | 0.225 | 0.12 | 0.545 |
| 655 | 1.142 | 0.024 | 0.248 | 0.136 | 0.662 |
| 660 | 1.407 | 0.022 | 0.278 | 0.22 | 0.785 |
| 663 | 1.526 | 0.025 | 0.296 | 0.247 | 0.83 |
| 665 | 1.614 | 0.026 | 0.3 | 0.268 | 0.879 |
| 670 | 1.538 | 0.027 | 0.287 | 0.278 | 0.835 |
| 675 | 1.193 | 0.021 | 0.25 | 0.222 | 0.613 |
| 680 | 0.65 | 0.008 | 0.195 | 0.12 | 0.378 |
| 685 | 0.398 | -0.009 | 0.154 | 0.073 | 0.185 |
| 690 | 0.186 | -0.016 | 0.118 | 0.021 | 0.083 |
| 695 | 0.073 | -0.016 | 0.104 | 0.06 | 0.027 |
| 700 | 0.073 | -0.025 | 0.098 | 0.022 | 0.04 |
| 705 | 0.016 | -0.029 | 0.092 | 0.026 | -0.005 |
| 710 | 0.011 | -0.032 | 0.091 | 0.026 | -0.009 |
| 715 | 0.008 | -0.031 | 0.088 | 0.026 | -0.01 |
| 720 | 0.009 | -0.029 | 0.089 | 0.024 | -0.01 |
Graph 2: The absorbance curve of different herbs according to spectrometer reading.
The result obtained from the experiment shows that the chlorophyll content is more in Brassica juncea with chlorophyll a 19.35g/gfw and chlorophyll b 16.88g/gfw and total chlorophyll content of a and b is 15.171 g/gfw, then followed by Tropaeolum majus with 10.52g/gfw of chlorophyll a and 0.949g/gfw and total of 8.98g/gfw chlorophyll, Opuntia sp. with 3.75g/gfw of chlorophyll a and 5.13g/gfw of chlorophyll b and total chlorophyll content with 4.58g/gfw, Rumex nepalensis with 3.13g/gfw of chlorophyll b and 2.73g/gfw of chlorophyll b and total chlorophyll content with 2.45g/gfw and lastly followed by Fagopyrum dibotrys 0.0037g/gfw of chlorophyll b and 0.707 g/gfw and total chlorophyll content of 0.6336g/gfw. The absorbance graph of chlorophyll content in the above specimen shows a similar pattern of chlorophyll content a and b with Brassica juncea with maximum absorbance followed by Tropaeolum majus, Opuntia sp., Rumex nepalensis and Fagopyrum dibotrys.
IV. Discussion
The chlorophyll content in the specimens vary greatly and Graph 2 also shows similar patterns as of chlorophyll content a and b. The chlorophyll content a and b is much more in the Brassica juncea than any other specimen in this experiment since the Brassica juncea was nurtured using proper nutrient supply in the garden with exposure of proper intensity of light and even the total amount of chlorophyll content was also high due to required nutrient was available for the plants growth. The chlorophyll a and b content in the Tropaeolum majus was also high apart from Brassica juncea since it has received a proper intensity of light but the nutrient was not supplied as it was grown in open space which makes low in chlorophyll content. It was proved that nutrients and minerals plays crucial role or directly proportional in developments of plants and amount of chlorophyll content in Eucalyptus plants by (Silva et al., 2014). The amount of chlorophyll a and b content in the Rumex nepalensis and Opuntia sp. are lower than Tropaeolum majus and Brassica juncea since it was receiving a partial sun rays and mostly grown in shade places and the soils are hardened with deprived of nutrients which makes low quantity of chlorophyll.
As per Olena et al. (2019), the amount of chlorophyll content is determined by the amount of sunlight that the plant receive, it is also influence by the shape and orientation of the leaves to the angle of incidence of light and the transmission of light through the leaves. When it comes to Fagopyrum dibotrys, the amount of chlorophyll a and b is very low since the plants was grown in shade and soil was hardened and deprived of nutrients. According to Gogoi & Basumatary (2018), they have found that the physical factors, soil nutrient, plant physiological and environmental plays a major role in amount of chlorophyll content and moreover the application of plant growth factors also plays a greater role in the production of the chlorophyll. The Graph 2 also shows amount of chlorophyll content is high in Brassica juncea, followed by Tropaeolum majus, R. nepalensis, Opuntia sp. and F. dibotrys due to the above mention reasons.
V. Conclusion
From the experiment it is conclude that the amount of chlorophyll content is high in Brassica juncea, followed by T. majus, R. nepalensis, Opuntia sp. and F. dibotrys. The amount of chlorophyll content depends on the amount of the nutrients available for the plants and amount of sunlight that they receive from the sun. Its shows that the plants that has more available with nutrient contains higher amount of chlorophyll (Brassica juncea) and plants that receives less sunlight and deprived of minerals and nutrients are with low content of chlorophyll. To have more correct and accurate result, the plants orientation, habitats of plants and nature of the plants should be taken into consideration by the future potential researchers.
VI. PRECAUTIONS
- The leaves midrib should be removed properly.
- The amount of leaves taken should not be more than 1g to avoid wrong reading
- The spectrometer should be operated 20 minutes early to have a correct reading.
- The measurements of reagents and chemical should be done properly to have correct reading.
- While preparing the extract, it should not be exposed to the sunlight.
- The acetone should be prepared 80% concentration and should be applied exact amount.
- The equipment’s and the spectrometer should be handle with care.
- After inserting the cuvette in the spectrophotometer, the lid should be covered properly.
VII. REFERENCES
Asimovic et al. (2016). Spectrophotometric determination of total chlorophyll content in fresh vegetables. 4.
Gogoi, M., & Basumatary, M. (2018). Estimation of the chlorophyll concentration in seven Citrus species of Kokrajhar district , BTAD , Assam , India. Tropical Plant Research, 5, 4. https://doi.org/10.22271/tpr.2018.v5.i1.012
Johan, F., Jafri, M. Z., Lim, H. S., & O, W. M. W. (2014). Laboratory measurement : Chlorophyll-a concentration measurement with acetone method using spectrophotometer. 4. https://doi.org/10.1109/IEEM.2014.7058737
Olena, V., Dzhamal, R., Oksana, S., & Valentyna, F. (2019). Estimation of photosynthetic pigments in the leaves of Crambe spp. during vegetation. Agrobiodiversity, 12. https://doi.org/https://doi.org/10.15414/agrobiodiversity.2019.2585-8246.373-381
Pareek et al. (2017). Chlorophylls : Chemistry and Biological Functions. 1–15.
Pareek et al. (2018). Chlorophylls : Chemistry and Biological Functions. Fruit and Vegetable Phytochemicals, 1, 16.
Shim, H. (2021). 2.1.5: Spectrophotometry. 1–5.
Silva, I., Ferreira, E., Pereira, I., Goncalves, J., Silva, E., Veloso, R., & Laia, M. (2014). Growth , Chlorophyll Content and Photosynthetic Capacity of Eucalyptus Clones Under Nutrition Omission. Australian Journal of Basic and Applied Sciences, 1–7.
