Absorption and action spectrum

Absorption and Action spectrum in Biophysics

1. Absorption and Action Spectrum 192bo001 S Narthanaa Ii Msc botany Kongunadu arts and science college Coimbarore [email protected]

2. Light The simplest definition of light is “ visually perceived radiant energy". “Visible” light is just a small segment of the electromagnetic spectrum— a broad range of radiant energy which also includes X-rays, ultraviolet and infrared energy, micro-waves and radio waves.

3. Spectrum The spectrum   is the range of  different  colours which is produced when light  passes  through a  glass  prism or through a  drop  of water. A  rainbow   shows  the colours in the spectrum.

4. Absorption of light Light absorption is a process by which light is absorbed and converted into energy . This process is known as photosynthesis in plants . However, light absorption doesn’t occur exclusively in plants , but in all creatures/inorganic substances. Absorption depends on the electromagnetic frequency of the light and object’s nature of atoms . Absorption of light is therefore directly proportional to the frequency . If they are complementary , light is absorbed . If they are not complementary , then the light passes through the object or gets reflected . Light absorption is related to matter's frequency (and frequency of light also) and wavelength of light.

5. The white light hits the ladybird's surface . The white light has all the colours of light, but when it hits the red surface , only the red light is reflected . The other colours are absorbed by the red surface. This means that when we look at the red parts of the ladybird, we only get red light reflected into our eyes. Therefore, when this reflected light hits our retina and the electrical impulse is sent to our brains, we see the red colour.

6. History J. B. L. Foucault In 1849, J. B. L. Foucault experimentally demonstrated that  absorption  and emission lines appearing at the same wavelength are both due to the same material, with the difference between the two originating from the temperature of the light source. T. W. Engelmann {Theodor Wilhelm} The first action spectrum was made by T. W. Engelmann, in 1882 who split light into its components by the prism and then illuminated Cladophora placed in a suspension of aerobic bacteria. He found that bacteria accumulated in the region of blue and red light of the split spectrum.

7. Lets see some facts Green plants are green because they contain a pigment called  chlorophyll as in the  absorption spectra , chlorophyll absorbs light in the red (long wavelength) and the blue (short wavelength) regions of the visible light spectrum. The green light is not absorbed but reflected , making the plant appear green .  An absorption spectrum shows all the light typically absorbed by a leaf. An action spectrum , meanwhile, shows all the light that is actually used for photosynthesis.  The similarity of the action spectrum of photosynthesis and the absorption spectrum of chlorophyll tells us that chlorophyll is the most important pigments in the process. The spectra are not identical, though, because carotenoid, which absorbs strongly in the blue , play a role as well.

8. Absorption Spectra An absorption spectrum is a spectrum of radiant energy whose intensity at each wavelength is a measure of the amount of energy at that wavelength that has passed through a selectively absorbing substance. The absorption of radiation by a substance can be quantified with an instrument called a  spectrophotometer. This is a device that produces a beam of monochromatic ("single-color") radiation that can be shifted progressively across the spectrum; P asses the beam through a solution of the substance, and measures the radiation that gets through.

9. The graph shows the absorption spectrum of a mixture of  chlorophyll a and chlorophyll b  in the range of visible light. .

10. Note that both chlorophylls absorb light most strongly in the red and violet portions of the spectrum. Green light is poorly absorbed so when white light (which contains the entire visible spectrum) shines on leaves, green rays are transmitted and reflected giving leaves their green color . The similarity of the action spectrum of photosynthesis and the absorption spectrum of chlorophyll tells us that chlorophylls are the most important pigments in the process. The spectra are not identical, though, because  carotenoids , which absorb strongly in the blue, play a role as well. The carotenoids help fill in the absorption gaps of chlorophyll so that a larger part of the sun's spectrum can be used. The energy absorbed by these   "antenna pigments"  is passed to chlorophyll a where it drives the light reactions of photosynthesis.

11. Case studies Beer's Law Plot For dilute solutions , the amount of light absorbed at a specific wavelength is directly proportional to the concentration of the solution. This relationship is called Beer's Law . A = absorbance (no units) ε = molar absorptivity coefficient (units = L/mol-cm) C = concentration of absorbing species (units = mol/L) l = path length (units = cm) A Beer’s Law Plot is a calibration curve of absorption plotted as a function of concentration. An absorption spectrum must be acquired first to determine the wavelength of maximum absorbance, λmax , for the compound being studied. All absorbances are acquired at this wavelength setting because the signal is the strongest and least likely to be obscured by instrument fluctuations. To create the plot, the absorbances of at least three solutions of known concentration are measured. A graph of absorbance versus concentration is constructed and a best fit straight line is drawn through the data points. Then the absorbance of a solution of unknown concentration is measured and its concentration is determined by comparison to the Beer's Law Plot. The “Plot” must be used in two ways to determine the unknown’s concentration. A = ε C l

12. Action Spectra An action spectrum is the rate of a physiological activity plotted against wavelength of light . In 1881, the German plant physiologist T. W. Engelmann placed a filamentous  green alga  under the microscope and illuminated it with a tiny  spectrum of visible light .

13. In the medium surrounding the strands were motile,   aerobic  bacteria . After a few minutes , the bacteria had congregated around the portions of the filament illuminated by red and blue light Assuming that the bacteria were congregating in regions where oxygen was being evolved in  photosynthesis , Engelmann concluded that red and blue light are the most effective colors for photosynthesis. With modern instruments, a plot of the rate of photosynthesis as a function of wavelength of light produces a graph like this. More precise than Engelmann's but telling the same story . In a 1972 paper, KJ McCree found that the action spectra for 22 different plant species were very similar. All showed that photosynthetic active radiation (PAR ) was between 400 nm and 700 nm and that all wavelengths within this region were used in photosynthesis.

14. Relation between absorption and action spectrum Pigments absorb light as a source of energy for photosynthesis The  absorption spectrum  indicates the  wavelengths of light absorbed  by each pigment (e.g. chlorophyll) The  action spectrum  indicates the  overall rate of photosynthesis  at each wavelength of light . There is a strong correlation between the cumulative absorption spectra of all pigments and the action spectrum Both display two main peaks – a larger peak at the blue region (~450 nm) and a smaller peak at the red region (~670 nm)  Both display a trough in the green / yellow portion of the visible spectra (~550 nm)

15. Differentiate between absorption spectrum and action spectrum Absorption Spectrum  The absorption spectrum indicates the wavelengths of light absorbed by each pigment (e.g. chlorophyll ) It is a graphical representation of the amount of light of different wavelengths absorbed by a pigment . It is studied directly . It requires a single exposure to full light . Action Spectrum  The action spectrum indicates the overall rate of photosynthesis at each wavelength of light It is a graphical representation of the rate of photosynthesis at different wavelengths of light . It is studied in relation to either O2​ evolved or CO2​ absorbed . It requires study of light utilization at various wavelengths

16. Spectrophotometer A spectrophotometer is an instrument that measures the amount of photons (the intensity of light) absorbed after it passes through sample solution. With the spectrophotometer, the amount of a known chemical substance (concentrations) can also be determined by measuring the intensity of light detected.

17. Single Beam UV-VIS Spectrophotometer

18. Applications Absorption Spectrum Absorption spectroscopy is useful in chemical analysis because of its specificity and its quantitative nature . Remote sensing : In atmospheric chemistry, differential optical absorption spectroscopy ( DOAS ) is used to measure concentrations of trace gases . Astronomical spectroscopy  is a particularly significant type of remote spectral sensing. Absorption spectroscopy is also employed in the study of  extrasolar planets . Absorption spectrum observed by the Hubble Space Telescope The infrared absorption spectrum of NASA laboratory sulfur dioxide ice is compared with the infrared absorption spectra of ices on Jupiter's moon, Io credit NASA, Bernard Schmitt, and UKIRT.

19. Action spectrum Action Spectrum for Phototropism The action spectra for single exposure UVR-induced  melanogenesis  and  erythema  in ‘fair’ untanned human skin The action spectrum of  photoinhibition  was first measured in vitro in 1966 (Jones and Kok , 1966 ). In   plant ecology , action spectra can be used for the practical purpose of predicting the biological response to be expected in a given situation.

20. Researches in this field Influence of Concentration on the Absorption Spectrum and the Action Spectrum of Fluorescence of Dye Solutions J . Lavorel - Cite this:  J. Phys. Chem.  1957, 61, 12, 1600–1605-Publication Date:December 1, 1957 https:// doi.org/10.1021/j150558a006

21. On the action spectra of photosynthesis and spectral dependence of the quantum efficiency            January 2000                 

22. The Photosynthetic Action Spectrum of the Bean Plant

23. Summary The action spectrum refers to the wavelengths of electromagnetic radiation (i.e. light) that are most effective for photosynthesis . An absorption spectrum is made up of the wavelengths of electromagnetic radiation (i.e.) light that are absorbed by a material they pass through. Maximum absorption  of light by chlorophyll a occurs in red and blue regions of the absorption spectrum. The absorption spectra of chlorophylls a and b (Fig. 13.11) show that they absorb maximum light in the blue-violet and red wavelengths. The pigments are often known after the wavelength which is absorbed to the maximum, e.g., Chl a 673  Chl a 683  (P 680 ), Chl a 703  (P 700 ). The graphic curve depicting the relative rates of photosynthesis at different wavelengths of light is called action spectrum (Fig. 13.11 B). It shows that maximum photo-synthesis occurs in blue-violet and red parts of the light.