Determination of Absorbance Spectra of Photosynthetic Pigments

Materials and Methods

The experimental procedure followed is set out in detail in the lab manual (School of Biological Sciences, 2005b). 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.

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. Literature values for the two light absorption peaks of chlorophyll a and b, respectively, are 420 nm and 435 nm for the first peak and 663 nm and 645 nm for the second peak.  Our results are close to the literature values and support the known fact that pigments do not absorb all wavelengths of light equally. There are two narrow ranges in the violet and red areas of the visible wavelength of light where chlorophyll a has the highest reading for absorbance and large amounts of photons are captured in those ranges. Similarly chlorophyll b has a specific spectrum shifted slightly more toward the green area of visible light where it can absorb the photons that chlorophyll a cannot. This results in the rate of photosynthesis remaining relatively high throughout the wavelength range, as shown in the action spectrum. Photon capture is more effective as a result of this varying absorbance at varying wavelength, thus meaning that plants can produce more energy through photosynthesis. The hypothesis formed, which was that the absorbance of light by chlorophyll a would not be equal to the absorbance by chlorophyll b at a wavelength of 480nm, therefore should have been shown to be true. 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.

References

Frigaard et al. (1996) Spectrochromatography of photosynthetic pigments as a fingerprinting technique for microbial phototrophs. FEMS Microbiol. Ecol. 20: 69-77

Knox B., Ladiges P., Evans B., and Saint R., (2005). Biology, An Australian focus, 3rd edition. McGraw Hill, pp.114-119

 School of Biological Sciences (2005a). BIOL1101 Laboratory and Lecture Notes, The University of Sydney, Sydney.

School of Biological Sciences (2005b). BIOL1101 Skills Book for Undergraduates, The University of Sydney, Sydney.

Appendix

1. Calculating t-Test

 Step One – Preliminary Calculations

Step Two – Calculating the t-test Statistic

SSa = ∑Xa^2 – (∑Xa)^2 / na = 0.0041 – (0.110)^2 / 4 = .0011

SSb = ∑Xb^2 – (∑Xb)^2 / na = 0.0370 – (0.243)^2 / 4 = .0222

S^2 = (SSa + SSb) / (na-1) + (nb-1) = (.0011 + .0222) / (3) + (3) = .0039

t = mean of a – mean of b / √S^2 (na+nb)/(na)(nb) = 0.030-0.090/√.0039(4+4)/(4)(4)

          = - 1.359

d.f. = 4-1+4-1 = 6

Critical value =2.447

2. Absorbance Values at 480nm

3. Table of Class Results

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