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Determination of Absorbance Spectra of Photosynthetic Pigments

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Introduction

Determination of Absorbance Spectra of Photosynthetic Pigments Abstract This experiment tested the hypothesis that the absorbance of light by chlorophyll a would not be equal to the absorbance by chlorophyll b at a wavelength of 480nm. Following extraction of lipid soluble pigments from silver beet by acetone and petroleum ether and separation via chromatography, the light absorbance of the individual pigments at various wavelengths was determined by spectrophotometry. Light absorption by chlorophyll a was observed to peak at 420nm and 660nm while that of chlorophyll b peaked at 440 nm and 600nm. Mean values for class results indicated that chlorophyll b (0.09) absorbed twice the amount of photons as did chlorophyll a (0.03) at a wavelength of 480nm. Since the results of t-testing gave a value (1.359) lower than the critical t- value (2.447), the results suggest that absorption values for chlorophyll a and b at 480nm were not significantly different, thus supporting acceptance of the null hypothesis. However, since our results are similar to reported literature values and support the literature documented fact that pigments do not absorb all wavelengths of light equally, it is believed that either faulty experimental technique or human error may have influenced the results obtained. ...read more.

Middle

Lipid-soluble pigments were extracted from silver beet by acetone and petroleum ether and deionised water was added. The individual pigments were then separated via chromatography and their absorbance at various wavelengths was determined using a spectrophotometer. The class results were then compiled and used to calculate mean values which were then discussed. Results At a wavelength of 480nm, chlorophyll b absorbed twice the amount of photons as did chlorophyll a. The following results are the mean absorbance values for chlorophyll a and chlorophyll b. Wavelength(nm) Chlorophyll a Chlorophyll b 480 0.030 0.090 T -testing resulted in a t-value of 1.359 and a critical t-value of 2.447 (see calculations in Appendix). As the calculated t-value was lower than the critical t-value, this fact suggested that the absorption values for chlorophyll a and b at 480nm were not significantly different, thus supporting acceptance of the null hypothesis. Discussion The results obtained showed that light absorption by chlorophyll a peaked at 420nm and 660nm while that of chlorophyll b peaked at 440 nm and 600nm. ...read more.

Conclusion

However due to the fact that our t-value was lower than that of our critical value the null hypothesis is suggested to be true and does not support our conclusion. This is cbelieved to be due to mistakes made in the method. The absorbance values for chlorophyll a and b at a wavelength of 480nm vary quite significantly in our replicates (see Appendix). This suggests that the conclusion of our t-test is quite likely due to error. This error could have come from various aspects of the experiment. While chlorophyll a and chlorophyll b possess a very similar chemical structure, chlorophyll a contains a methyl group whereas chlorophyll b contains a formyl group (Frigaard et al., 1996). This causes chlorophyll a to be less polar than chlorophyll b and thus they are not equally eluted from the chromatography matrix into the acetone. Therefore the concentrations of chlorophyll a and b are not equal and will affect the absorbance readings, as the more concentrated substance will thus be more absorbent. It is also possible that the absorbance readings were not exact as it was often difficult to read the spectrophotometer although there is no direct proof that human error has resulted in faulty results. ...read more.

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