Using Spectroscopy to Evaluate the Absorption of Light for Different Substances
Using Spectroscopy to Evaluate the Absorption of Light for Different Substances
Laura and Polina, Section 13
BIOL 130, Monday 7:00-9:50pm Rm B2 151
Performed on October 20th, 2008
The purpose of experiment one was to determine the concentration of the unknown solution by using spectroscopy and comparing that substance to substances of known concentrations. In experiment two, the purpose was to discover the regions of the visible light spectrum are absorbed by both samples of chlorophyll. The spectrophotometer was an exceptionally useful tool for this lab. Spectroscopy is valuable for identifying substances through absorption of light which is done by measuring substances and comparing them to other known substances. Specifically a spectrophotometer is a device that measures the absorption of radiation at a particular wavelength. This is done by a light bulb shining, refracting its light into one beam, which then passes through an exit slit, then through the test solution and to a detector. This detects the amount of light that passed through the substance and a readout shows the amount of light that was absorbed (Jones, A, et al, 2007).
Beer’s Law states that concentration of a substance is directly proportional to its amount of light absorption (Department of Biology, 2008). The number of molecules of the solute is related exponentially to the amount of light that is absorbed while passing through the solute, called the solute concentration. Using Beer’s Law with known absorptivity at the absorption maximum a substance’s concentration within a solute can be measured. Since spectrophotometers are constructed to give absorbance, concentration can be figured out through their relationship to each other (Jones, A, et al, 2007).
Considering the fact that distilled water was used as a blank and also used to dilute the solutions, the output of the absorbance of the different solutions is compared to distilled water. The concentration of distilled water should be zero, meaning the absorbance of light should be zero as well (Jones, A, et al, 2007).
Experiment one used the substance fast green which is a dye. In experiment number two the substance used was chloroplast pigment (Department of Biology, 2008).
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Chloroplasts are organelles located in mesophyll cells of leaves. They have DNA and are self replicating. These organelles are where the process of photosynthesis takes place, making plants semi self sufficient. In these chloroplasts are chlorophylls which are molecules or pigments which capture the light from the sun. Chlorophylls only absorb certain wavelengths of light and reflect certain wavelengths. The wavelengths they reflect are green in colour which is why leaves are green (Karp, 2008).
Materials and Methods
All steps were carried out as stated in the lab manual. The only deviation that occurred was that when the absorbance reached a peak, the absorption was not measured at 5 nm increments, non-intentionally. (pages 34-40 of the BIOL 130 Fall 2008 Lab Manual)
The results for the absorbance of fast green in cuvette 1, the stock solution, are shown in Table 1. This data shows that there is a peak of absorption at the wavelength 620nm.
The results for the absorptions of cuvettes #1-5 at the peak wavelength of 620nm is shown in Table 2. This data shows that a decrease in concentration results in a decrease in absorbance.
The results for the absorption of light for cholorphyll A and B are presented in Table 3. The peaks of absorption for chlorophyll A are 430nm, 615nm and 665nm. The peaks for chlorophyll B are 455nm, 600nm, 645nm.
Calculations for Concentration
Concentration in cuvette #1 – stock solution – 0.015mg/mL
Concentration in cuvette #2 – C1V1=C2V2
Concentration in cuvette #3 – using same calculation – 0.00375mg/mL
Concentration in cuvette #4 – using same calculation – 0.001875mg/mL
Concentration in cuvette #5 – using same calculation – 0.0009375mg/mL
Figure 1: Absorption Spectrum for Fast Green
Absorption Spectrum for Fast Green
The results of the absorption spectrum for the stock solution of fast green are displayed in Figure 1. This shows that the maximum absorption of light for fast green is at a wavelength of 620nm.
Figure 2: Concentration Curve of Fast Green
Concentration Curve of Fast Green
The results of the concentrations of the different concentrations of fast green are shown in Figure 2. This is an approximately linear distribution of the data and can be used to figure out the approximate concentration of the unknown.
Figure 3: Drawing of Chromatograph for Chloroplast
The results for the chromatograph of the chloroplast are shown in Figure 3. This shows the separation of the four different substances in the chloroplast. The orange is band is carotene, the two yellow bands are xanthophylls and the light green band followed by a dark green band is chlorophyll A and B respectively (Department of Biology, 2008).
Figure 4: The Absorption Spectrum for Chlorophyll A and B
Absorption Spectrum for Chlorophyll A and B
The results of the absorption spectrum for both chlorophyll A and B are shown in Figure 4. This data shows that chlorophyll A and B both have major peaks and chlorophyll A has the major peak at a lower wavelength than chlorophyll B.
This experiment used spectroscopy as a means to find the light absorption and in turn the absorption spectrum for the substances fast green and chlorophyll A and B. To begin, the fast green was a dark green colour, chlorophyll A was a green-yellow colour and chlorophyll B was a true light green colour. When diluted, the fast green had visually changed to a paler shade of green and was more transparent than before. If we visually see a colour that is more pale it means that it is reflecting more light and absorbing less. This means that the expected result of fast green when diluted is that it will absorb less light. The actual result matched these expected results and as the concentrations of the fast green solutions lessened, so did the amount of light absorbed as shown in Table 2. There also had to have been a peak absorption of light because that is the light of the colour that we see the least of. As shown in Appendix I, Figure 5, the peak of 620nm, as shown in Table 1 and Figure 1, is the orange colour. This means we don`t see much orange colour in the fast green because it is absorbed instead of reflected. The colour of the light that is the most reflected is the green-blue colour.
The concentration for the unknown # 124 was about 0.0064mg/mL according to Figure 2. The concentration curve for fast green was used to figure out the concentration of the unknown, by using the found absorption at the peak wavelength. The absorption for the unknown was 0.04 and when compared to the fast green concentration curve the unknown concentration was determined as the above. This works because it is a linear distribution of the data that states if the concentration of the substance increases, then the absorption of the substance increases. However, this concentration may not be very accurate due to the graphing discrepancies.
When the chloroplast pigment was used in chromatography, it was split into its components, two of which were chlorophyll A and chlorophyll B, as shown in Figure 3. The absorption spectrums for each are different because chlorophyll A is a lighter colour than chlorophyll B, as shown in Figure 4 and Figure 6. This absorbance spectrum for chlorophyll A and B are quite accurate to show what colour of light is reflected and what is absorbed when compared to the actual absorbance spectrum, Figure 5. According to the spectrum, chlorophyll A reflects more blue and yellow light, and chlorophyll B reflects more green and yellow light. This is quite accurate because as shown on the chromatograph these are approximately the colours that are shown.
The sources of error in this lab are numerous. Some of the major sources of error that could have had an effect on the concentration of the unknown are the volume of distilled water and fast green when diluting may have been incorrect, making the unknown have a higher concentration if the fast green had a higher concentration. The same can be said for lower concentration. The major source of error in this lab is that the fast green diluted substance was not measured at 5nm wavelength intervals when there was a peak and therefore the unknown substance could not have been measured at the correct wavelength. This error could have the unknown’s concentration be higher or lower than it actually is depending on whether the actual wavelength peak was higher or lower than 620nm.
The sources of error for the chloroplast chromatography are that the line of the pigment, when applying a second coat the first coat was not dry. This could have affected the separation and not separated the chlorophylls enough and therefore affected the absorption spectrum making it seem like it reflected different colours than it actually did. Also, the chlorophyll was not in the acetone for very long and could not have had long enough to fully dispense the entire colour from the chromatograph paper.
The entire experiment could have been wrong if the cuvettes were not facing the right way every time. Because of the difference in the thickness of the glass on different sides of the cuvette, the light absorption can be skewed for each wavelength.
Overall, this experiment was successful and determined the concentration of an unknown and created an absorption spectrum for chlorophyll A and B.
Figure 5: The actual absorption spectrum of chlorophyll A and B
(Karp, 2008) pp. 220
Figure 6: Actual Picture of Chromatograph of Chloroplast (taken in class)
Department of Biology 2008 Introductory Cell Biology Laboratory Manual. University of
Waterloo, Waterloo. pp. 34-40.
Jones, A, et al. 2007. Practical Skills in Biology Fourth Edition. Pearson Education Limited.
London. pp. 366-369.
Karp, G. 2008. Cell and Molecular Biology Concepts and Experiments, Fifth Edition. John
Wiley & Sons, Inc. Hoboken. pp. 216-221.