Question
BI 335- LAB 2- ASSESSED PRACTICAL
Finding the Absorption Spectrum of Coleus Leaves
Plants contain primary and secondary pigments that are necessary for absorbing energy
utilized in the process of photosynthesis. Students will determine the absorption spectrum of a
sample to derive the wavelengths most efficiently used by the pigments in different colored
leaves.
Advance Preparation: Turn on the spectrophotometer to allow warm-up time to assure
accuracy in measurement.
Part A: Sample Preparation
Live plant material is used in the absorption analysis. Chlorophyll and other pigments are
removed using the protocol provided below.
Materials (per team):
Balance
Coleus leaf
Mortar and pestle
30 mL of ethanol
35 mL syringe
100 mL beaker
Funnel lined with a coffee filter
Safety goggles
SAFETY
Wear goggles and aprons when working with chemicals.
Alcohol is volatile and flammable.
Avoid breathing in vapors of the reagents.
Procedure:
1. Prepare an extract of chlorophyll by weighing 0.3 grams of your leaf sample. Make sure the
sample includes all of the different colors on the leaf. Tear the leaf section into small pieces
and place the tissue in a mortar for grinding.
2. Add 10 mL of ethanol to the sample and grind with the pestle until only tiny specks of leaf
tissue remain. Add an additional 20 mL of ethanol to the mortar.
3. Filter this liquid into a beaker through a funnel lined with a coffee filter.
4. Dispose of the filter in the designated trash bin.
Part B: Calibration
The extract must fall into a specified range to provide a reading with the spectrophotometer. A
sample that is too concentrated will not provide a valid measurement. To assure the sample
falls within a readable concentration range, follow the steps provided.
Materials:
Two 3 mL syringes
Ethanol
Plant extract from Part A
2 cuvettes
Kimwipes
Sharpie marker
Small test tube rack
Spectrophotometer or Calorimeter.
Procedure:
1. Set the spectrophotometer wavelength to 640 nm.
2. Push the A/T/C button to select % transmittance. The digital readout will show %T.
3. Label one of the 3 mL syringes ethanol. Label the second syringe leaf extract.
4. Dispense 3 mL of ethanol into a clean glass cuvette. (Wipe off finger prints with a Kimwipe.)
Important: This cuvette is a “blank” that will be used throughout this lab to calibrate the
spectrophometer.
5. Insert the blank in the cell holder and close the door.
6. Press the 0 ABS/100%T key to set the blank to 100% transmittance.
7. The blank provides a standard for reading. Remove the blank and proceed with testing the
extract by following the same procedure.
8. Dispense 3 mL of the extract into a clean glass cuvette. (Wipe off all fingerprints with a
Kimwipe.)
9. Insert the cuvette in the cell holder and close the door.
10. A reading of 30%-40% transmittance is best. If %T is greater than 40%, you may skip the
dilution step. If it shows less than 30% transmittance, dilution of the sample is required.
11. To dilute the filtered extract, add 0.5 mL of ethanol (using the clean ethanol syringe).
Tightly hold a finger over the cuvette and invert to mix. Re-test the
percent transmittance. Continue with 0.5 mL increments of ethanol until the reading is greater
than 30% transmittance.
Part C: Testing Plant Extract
Determine the absorption spectrum for the pigments in the coleus leaf following the steps
provided.
Materials:
Blank prepared in Part B
Plant extract from Part A
Kimwipes
Procedure:
1. Look at the data table and note the readings range from 400 nm – 700 nm (with increments
of 20nm). This is the range for testing.
2. Set the wavelength on the spectrophotometer to 400 nm.
3. Set the mode to “absorbance” by pressing the A/T/C button until an A appears at the right
of the digital display.
4. Insert the blank (clean with a Kimwipe) in the cell holder and close the door.
5. Press the 0 ABS/100%T key to set the blank to 0 absorbance.
6. Remove the blank and insert the clean cuvette containing the chlorophyll extract.
7. Record the digital readout for the absorbance in the data table.
8. Continue to increase the wavelength by 20 nm each time and repeat steps 4-7. Remember
to use the blank for each new wavelength setting!!
9. Graph the data using the instructions provided for Excel 2007.
10. Dispose of the leaf extract in the waste container in the fume hood.
Result Collected from the experiment are in the table below;
Wavelength: Absorbance: Percentage:
450nm 1.681 (Violet) 2.1%
500nm 0.631 (Blue) 23.4%
550nm 0.39 (Green) 40%
570nm 0.407 (Yellow) 39.1%
600nm 0.485 (Orange) 32.7%
650nm 0.685 (Red) 20.7%
Refer to the Lab Practical 2 above that contain Materials, Procedure, Instruction, Results, etc, and Provide a full Lab Report Write up that must contain, Title, Aim, Introduction, Materials, Procedure, Results, Discussion, Conclusion, Recommendation, and the References.
Finding the Absorption Spectrum of Coleus Leaves
Plants contain primary and secondary pigments that are necessary for absorbing energy
utilized in the process of photosynthesis. Students will determine the absorption spectrum of a
sample to derive the wavelengths most efficiently used by the pigments in different colored
leaves.
Advance Preparation: Turn on the spectrophotometer to allow warm-up time to assure
accuracy in measurement.
Part A: Sample Preparation
Live plant material is used in the absorption analysis. Chlorophyll and other pigments are
removed using the protocol provided below.
Materials (per team):
Balance
Coleus leaf
Mortar and pestle
30 mL of ethanol
35 mL syringe
100 mL beaker
Funnel lined with a coffee filter
Safety goggles
SAFETY
Wear goggles and aprons when working with chemicals.
Alcohol is volatile and flammable.
Avoid breathing in vapors of the reagents.
Procedure:
1. Prepare an extract of chlorophyll by weighing 0.3 grams of your leaf sample. Make sure the
sample includes all of the different colors on the leaf. Tear the leaf section into small pieces
and place the tissue in a mortar for grinding.
2. Add 10 mL of ethanol to the sample and grind with the pestle until only tiny specks of leaf
tissue remain. Add an additional 20 mL of ethanol to the mortar.
3. Filter this liquid into a beaker through a funnel lined with a coffee filter.
4. Dispose of the filter in the designated trash bin.
Part B: Calibration
The extract must fall into a specified range to provide a reading with the spectrophotometer. A
sample that is too concentrated will not provide a valid measurement. To assure the sample
falls within a readable concentration range, follow the steps provided.
Materials:
Two 3 mL syringes
Ethanol
Plant extract from Part A
2 cuvettes
Kimwipes
Sharpie marker
Small test tube rack
Spectrophotometer or Calorimeter.
Procedure:
1. Set the spectrophotometer wavelength to 640 nm.
2. Push the A/T/C button to select % transmittance. The digital readout will show %T.
3. Label one of the 3 mL syringes ethanol. Label the second syringe leaf extract.
4. Dispense 3 mL of ethanol into a clean glass cuvette. (Wipe off finger prints with a Kimwipe.)
Important: This cuvette is a “blank” that will be used throughout this lab to calibrate the
spectrophometer.
5. Insert the blank in the cell holder and close the door.
6. Press the 0 ABS/100%T key to set the blank to 100% transmittance.
7. The blank provides a standard for reading. Remove the blank and proceed with testing the
extract by following the same procedure.
8. Dispense 3 mL of the extract into a clean glass cuvette. (Wipe off all fingerprints with a
Kimwipe.)
9. Insert the cuvette in the cell holder and close the door.
10. A reading of 30%-40% transmittance is best. If %T is greater than 40%, you may skip the
dilution step. If it shows less than 30% transmittance, dilution of the sample is required.
11. To dilute the filtered extract, add 0.5 mL of ethanol (using the clean ethanol syringe).
Tightly hold a finger over the cuvette and invert to mix. Re-test the
percent transmittance. Continue with 0.5 mL increments of ethanol until the reading is greater
than 30% transmittance.
Part C: Testing Plant Extract
Determine the absorption spectrum for the pigments in the coleus leaf following the steps
provided.
Materials:
Blank prepared in Part B
Plant extract from Part A
Kimwipes
Procedure:
1. Look at the data table and note the readings range from 400 nm – 700 nm (with increments
of 20nm). This is the range for testing.
2. Set the wavelength on the spectrophotometer to 400 nm.
3. Set the mode to “absorbance” by pressing the A/T/C button until an A appears at the right
of the digital display.
4. Insert the blank (clean with a Kimwipe) in the cell holder and close the door.
5. Press the 0 ABS/100%T key to set the blank to 0 absorbance.
6. Remove the blank and insert the clean cuvette containing the chlorophyll extract.
7. Record the digital readout for the absorbance in the data table.
8. Continue to increase the wavelength by 20 nm each time and repeat steps 4-7. Remember
to use the blank for each new wavelength setting!!
9. Graph the data using the instructions provided for Excel 2007.
10. Dispose of the leaf extract in the waste container in the fume hood.
Result Collected from the experiment are in the table below;
Wavelength: Absorbance: Percentage:
450nm 1.681 (Violet) 2.1%
500nm 0.631 (Blue) 23.4%
550nm 0.39 (Green) 40%
570nm 0.407 (Yellow) 39.1%
600nm 0.485 (Orange) 32.7%
650nm 0.685 (Red) 20.7%
Refer to the Lab Practical 2 above that contain Materials, Procedure, Instruction, Results, etc, and Provide a full Lab Report Write up that must contain, Title, Aim, Introduction, Materials, Procedure, Results, Discussion, Conclusion, Recommendation, and the References.
Answers
GPT-4o mini
# Lab Report: Finding the Absorption Spectrum of Coleus Leaves
## Title
Determining the Absorption Spectrum of Coleus Leaves to Identify the Wavelengths Most Efficiently Used by Pigments in Photosynthesis
## Aim
The aim of this experiment is to extract pigments from Coleus leaves and determine their absorption spectrum in the range of 400 nm to 700 nm to identify which wavelengths are most efficiently used by the pigments during the process of photosynthesis.
## Introduction
Photosynthesis is a critical process by which plants convert light energy into chemical energy in the form of glucose. The efficiency of photosynthesis greatly depends on the pigments present in the plant leaves, primarily chlorophylls and carotenoids. Chlorophyll a and b absorb light most efficiently in the blue-violet and red parts of the spectrum, while carotenoids absorb mainly in the blue and green areas. The goal of this practical is to extract the pigments from Coleus leaves and observe their absorption characteristics across a defined spectrum using a spectrophotometer. This data can be used to infer which colors of light are most useful in driving the photosynthetic process.
## Materials
- Balance
- Coleus leaf (including various colors)
- Mortar and pestle
- Ethanol (30 mL)
- 35 mL syringe
- 100 mL beaker
- Funnel lined with a coffee filter
- Safety goggles
- Spectrophotometer
- Two 3 mL syringes
- Kimwipes
- Sharpie marker
- Small test tube rack
- Cuvettes (2)
## Procedure
### Part A: Sample Preparation
1. Weigh 0.3 grams of Coleus leaf material ensuring it contains various colors.
2. Tear the leaf into small pieces and place it into the mortar. Add 10 mL of ethanol and grind with the pestle until only small specks of tissue remain.
3. Add an extra 20 mL of ethanol to the mortar and mix thoroughly.
4. Filter the liquid using a funnel lined with coffee filter into a beaker, discarding the leaf material.
### Part B: Calibration
1. Set the spectrophotometer wavelength to 640 nm.
2. Select % transmittance mode using the A/T/C button until the display shows %T.
3. Label one syringe as 'ethanol' and the second as ‘leaf extract’.
4. Fill the first cuvette with 3 mL of ethanol to create a blank, wiping all fingerprints with a Kimwipe.
5. Place the cuvette in the spectrophotometer and calibrate it by pressing the 0 ABS/100%T key.
6. Remove the blank and fill the second cuvette with 3 mL of the leaf extract, wiping it clean before insertion.
7. Record the % transmittance. If it exceeds 40%, proceed. If below 30%, dilute by adding 0.5 mL of ethanol and retest until readings are appropriate.
### Part C: Testing Plant Extract
1. Adjust the spectrophotometer to measure from 400 nm to 700 nm in 20 nm increments.
2. For each wavelength:
a. Set the spectrophotometer to the selected wavelength.
b. Insert the blank cuvette, calibrate to 0 absorbance.
c. Replace with the cuvette containing the leaf extract and record the absorbance.
3. Continue this for wavelengths 400 nm to 700 nm.
### Results
| Wavelength (nm) | Absorbance | Percentage Transmittance |
|------------------|-------------|--------------------------|
| 450 | 1.681 | 2.1% |
| 500 | 0.631 | 23.4% |
| 550 | 0.390 | 40% |
| 570 | 0.407 | 39.1% |
| 600 | 0.485 | 32.7% |
| 650 | 0.685 | 20.7% |
## Discussion
The results indicate distinct absorbance properties of the pigments extracted from the Coleus leaves. Notably, the highest absorbance occurs around 450 nm, where chlorophyll a is most effective at trapping energy, primarily used in photosynthesis. The absorbance decreases progressively into the green (550 nm) and yellow (570 nm) range, correlating with the expected properties of chlorophyll, which reflects rather than absorbs green light—a key characteristic observed in many green plants.
In agreement with known data, the extract also suggests that while chlorophyll absorbs light efficiently in the blue and red regions, other pigments may be involved in absorbing wavelengths throughout the visible spectrum.
## Conclusion
The experiment successfully determined the absorption spectrum of pigments from Coleus leaves, revealing their absorbance characteristics across various wavelengths. Chlorophyll, as anticipated, shows significant absorbance peaks in the blue-violet (450 nm) and red (around 650 nm) regions. The findings underline the efficiency of these pigments in harnessing light for photosynthesis.
## Recommendations
Further experiments could include comparing the absorption spectra of different leaf types or analyzing the effects of varying light conditions on pigment extraction and absorption. Additionally, quantifying the concentrations of different pigments via chromatographic methods could enhance understanding of how various pigments contribute to overall absorption spectra and photosynthetic efficiency.
## References
1. Taiz, L., & Zeiger, E. (2010). Plant Physiology. 5th Edition. Sinauer Associates.
2. Raven, P. H., Graham, E. & Johnson, G. B. (2005). Biology. 7th Edition. McGraw Hill.
3. Evert, R. F., & Eichhorn, S. E. (2013). Raven Biology of Plants. 8th Edition. W.H. Freeman and Company.
## Title
Determining the Absorption Spectrum of Coleus Leaves to Identify the Wavelengths Most Efficiently Used by Pigments in Photosynthesis
## Aim
The aim of this experiment is to extract pigments from Coleus leaves and determine their absorption spectrum in the range of 400 nm to 700 nm to identify which wavelengths are most efficiently used by the pigments during the process of photosynthesis.
## Introduction
Photosynthesis is a critical process by which plants convert light energy into chemical energy in the form of glucose. The efficiency of photosynthesis greatly depends on the pigments present in the plant leaves, primarily chlorophylls and carotenoids. Chlorophyll a and b absorb light most efficiently in the blue-violet and red parts of the spectrum, while carotenoids absorb mainly in the blue and green areas. The goal of this practical is to extract the pigments from Coleus leaves and observe their absorption characteristics across a defined spectrum using a spectrophotometer. This data can be used to infer which colors of light are most useful in driving the photosynthetic process.
## Materials
- Balance
- Coleus leaf (including various colors)
- Mortar and pestle
- Ethanol (30 mL)
- 35 mL syringe
- 100 mL beaker
- Funnel lined with a coffee filter
- Safety goggles
- Spectrophotometer
- Two 3 mL syringes
- Kimwipes
- Sharpie marker
- Small test tube rack
- Cuvettes (2)
## Procedure
### Part A: Sample Preparation
1. Weigh 0.3 grams of Coleus leaf material ensuring it contains various colors.
2. Tear the leaf into small pieces and place it into the mortar. Add 10 mL of ethanol and grind with the pestle until only small specks of tissue remain.
3. Add an extra 20 mL of ethanol to the mortar and mix thoroughly.
4. Filter the liquid using a funnel lined with coffee filter into a beaker, discarding the leaf material.
### Part B: Calibration
1. Set the spectrophotometer wavelength to 640 nm.
2. Select % transmittance mode using the A/T/C button until the display shows %T.
3. Label one syringe as 'ethanol' and the second as ‘leaf extract’.
4. Fill the first cuvette with 3 mL of ethanol to create a blank, wiping all fingerprints with a Kimwipe.
5. Place the cuvette in the spectrophotometer and calibrate it by pressing the 0 ABS/100%T key.
6. Remove the blank and fill the second cuvette with 3 mL of the leaf extract, wiping it clean before insertion.
7. Record the % transmittance. If it exceeds 40%, proceed. If below 30%, dilute by adding 0.5 mL of ethanol and retest until readings are appropriate.
### Part C: Testing Plant Extract
1. Adjust the spectrophotometer to measure from 400 nm to 700 nm in 20 nm increments.
2. For each wavelength:
a. Set the spectrophotometer to the selected wavelength.
b. Insert the blank cuvette, calibrate to 0 absorbance.
c. Replace with the cuvette containing the leaf extract and record the absorbance.
3. Continue this for wavelengths 400 nm to 700 nm.
### Results
| Wavelength (nm) | Absorbance | Percentage Transmittance |
|------------------|-------------|--------------------------|
| 450 | 1.681 | 2.1% |
| 500 | 0.631 | 23.4% |
| 550 | 0.390 | 40% |
| 570 | 0.407 | 39.1% |
| 600 | 0.485 | 32.7% |
| 650 | 0.685 | 20.7% |
## Discussion
The results indicate distinct absorbance properties of the pigments extracted from the Coleus leaves. Notably, the highest absorbance occurs around 450 nm, where chlorophyll a is most effective at trapping energy, primarily used in photosynthesis. The absorbance decreases progressively into the green (550 nm) and yellow (570 nm) range, correlating with the expected properties of chlorophyll, which reflects rather than absorbs green light—a key characteristic observed in many green plants.
In agreement with known data, the extract also suggests that while chlorophyll absorbs light efficiently in the blue and red regions, other pigments may be involved in absorbing wavelengths throughout the visible spectrum.
## Conclusion
The experiment successfully determined the absorption spectrum of pigments from Coleus leaves, revealing their absorbance characteristics across various wavelengths. Chlorophyll, as anticipated, shows significant absorbance peaks in the blue-violet (450 nm) and red (around 650 nm) regions. The findings underline the efficiency of these pigments in harnessing light for photosynthesis.
## Recommendations
Further experiments could include comparing the absorption spectra of different leaf types or analyzing the effects of varying light conditions on pigment extraction and absorption. Additionally, quantifying the concentrations of different pigments via chromatographic methods could enhance understanding of how various pigments contribute to overall absorption spectra and photosynthetic efficiency.
## References
1. Taiz, L., & Zeiger, E. (2010). Plant Physiology. 5th Edition. Sinauer Associates.
2. Raven, P. H., Graham, E. & Johnson, G. B. (2005). Biology. 7th Edition. McGraw Hill.
3. Evert, R. F., & Eichhorn, S. E. (2013). Raven Biology of Plants. 8th Edition. W.H. Freeman and Company.