Photosynthesis in Non-Green Plants
Laboratory and Research skills Title: PRACTICAL 4&5 PHOTOSYNTHESIS IN NON-GREEN PLANTS Introduction Photosynthesis is a vital process that occurs in plants and is carried out by a sequence of pigments which have the ability to absorb visible light.
The most vital pigments are chlorophylls which are green in colour. From this it can be concluded that green plants contain chlorophyll.
However, plants of other colours, such as red, still have the ability to photosynthesis; therefore other pigments must be involved in the process of photosynthesis. The aim of this investigation was to determine whether or not there is a difference between the combinations of pigments in red leaves in comparison to those of green leaves. Hypothesis: Red plants do not possess the same combination of pigments as green plants Method One gram of fresh, torn green leaves (spinach) was weighed out. The leaves were then transferred to a test tube and 4.
mL of acidified methanol/acetone was added and the test tube was shaken until the methanol turned bright green in colour. The mixture was then filtered and collected in a new test tube.
This process was then repeated using a red leaf (coleus). Spectrophotometry 0. 5mL of the pigment sample was then transferred into a cuvette1. Readings from the spectrophotometer were obtained and recorded over an increasing wavelength range.
Thin Layer Chromatography (TLC) 18. 4mL of petroleum ether was poured into a beaker along with 1. mL of acetone; this was then transferred to the TLC tank. A line was drawn 1cm above the bottom of the TLC plate. 5µL – 15µL of pigment solution was drawn into dropper pipette and then applied to the TLC plate2: this process was carried out six times.
The TLC plate was then immersed in solvent solution and left, allowing the solvent to travel 10cm. The plate was then examined under a UV light and the distance travelled marked. Results Graph 1 Graph 2 Discussion Graph 1 shows results for the absorbance of the pigments after they had been dissolved in acetone.
Both the green leaf and the red leaf pigments appear to follow a very similar absorbance pattern. However, at 650nm the absorbance of the pigments in the green leaf rise sharply, it is likely that this is simply a discrepancy. It was concluded from this experiment that the solvent used was not the most effective.
For the next experiment the solvent was changed to acidified methanol. No results were obtained for the TLC plate as the pigments did not dissolve fully in the solvent, therefore could not travel up the plate and consequently we were unable to record any results.
Graph 2 illustrates the results obtained for the absorbance of the pigments after they had been dissolved in acidified methanol. The range for the spectrophotometer was increased, starting at a wavelength of 200nm instead of 400nm in an attempt to gain a clearer set of results. The results obtained were much more defined and allowed us to gain an understanding of the differing pigmentation between red and green leaves.
From the graph it can be seen that the green leaf pigments have a high absorbance at short wavelengths, peaking very early at 250nm – 350nm.
It was noticed that the red pigments did not give an absorbance reading for 200nm, 250nm or 300nm which highlighted a difference between the leaves. As the green pigment absorbance begins to decrease the red leaf pigment absorbance begins to rise and then peaks as the green pigments reach their lowest absorbance at 500nm. The red leaf pigments absorbance then decreases and eventually both lines meet. The TCL plate was examined under a UV light and some pigments could be seen.
However, no results were recorded accurate measurements could not be taken and most of the pigments had travelled off the end of the plate.
Figure 1: The absorption spectrum of pigments4 From the results obtained it can be concluded that red and green leaves possess different combinations of pigments, proving our hypothesis true. The main evidence to support this comes from Graph 2 where peaks for the green and red leaf occur at different wavelengths. Both colours of leaves contain accessory pigments which have the ability to absorb light energy and pass it on to a primary pigment3 which in turn commences the process of photosynthesis.
On comparing Graph 2 to Figure 1, it can be presumed that green leaves contain a greater concentration of the pigment Chlorophyll a whilst red leaves appear to have a higher concentration of Phycoerythrin, Chlorophyll b and carotenoids.
Both colours of leaves possess accessory pigments but red leaves have more. This is because photosynthesis occurs over the green section of the light spectrum and the red leaf absorbs different wavelengths of light compared green leaves.
In order to carry out photosynthesis successfully the red leaf requires a greater selection of accessory pigments which have the ability to absorb light over a range of wavelengths. Evaluation Initially acetone was used as the solvent and this affected the results for both spectrophotometry and TLC. The pigments did not dissolve successfully and for that reason they could not travel any distance along the TLC plate. Similarly for the spectrophotometry there was no clear differentiation between the pigments in either leaf.
The next time the experiment was carried out the solvent was changed from acetone to acidified methanol.
It was noticed almost immediately that the solutions were much brighter which indicated that the pigments were being dissolved out of the leaves much more successfully. The acidified methanol was a more effective solvent. The pigments dissolved and dispersed throughout the methanol which allowed for a more accurate reading from the spectrophotometer. Unfortunately the TLC plate was left for too long a period of time and the pigments travelled off the end of the plate, subsequently we were unable to take any measurements or obtain an Rf value.
For future experiments the most important alteration to be made would be to our TLC method.
Using acidified methanol as a solvent carry out the same process but ensure that the plate is not left for too long. This will allow for accurate readings and measurements to be taken. Appendix Table 1 – Absorbance of red and green leaves using acetone as the solvent | Absorbance| Wavelength (nm)| Green Leaf| Red Leaf| 400| 1. 472| 1. 521| 450| 1.
556| 1. 200| 500| 0. 301| 0. 445| 550| 0. 194| 0. 372| 600| 0.
370| 0. 471| 650| 0. 650| 0. 409| 700| 0. 80| 0. 110| 750| 0.
061| 0. 079| 800| 0. 049| 0. 058| 850| 0. 045| 0.
053| 900| 0. 033| 0. 046| 950| 0. 041| 0. 047| Table 2 – Absorbance of red and green leaves using acidified methanol as the solvent | Absorbance| Wavelength (nm)| Green Leaf| Red Leaf| 200| 0.
610| 0. 862| 250| 1. 807| -| 300| 1. 535| -| 350| 1. 512| -| 400| 0. 573| 0.
478| 450| 0. 301| 0. 375| 500| 0. 074| 1. 285| 550| 0.
006| 1. 140| 600| 0. 075| 0. 046| 650| 0. 120| 0.
027| 700| 0. 069| 0. 012| 750| 0. 048| 0. 010| 800| 0.
013| 0. 004| 850| 0. 011| 0. 003| 900| 0. 006| 0. 02| 950| 0.
007| 0. 005| References  Bonner PL & Hargreaves AJ Basic Bioscience Laboratory Techniques A pocket guide. West Sussex: John Wiley & Son, Ltd; 2011.  Bonner PL & Hargreaves AJ Basic Bioscience Laboratory Techniques A pocket guide. West Sussex: John Wiley & Son, Ltd; 2011.
 [homepage on the Internet]. 2009 [cited 2011 Oct 25]. Available from: http://hyperphysics. phy-astr. gsu.
edu/hbase/biology/pigpho. html  [image on the Internet]. [cited 2011 Oct 26]. Available from: http://http://bit. ly/tgrWJel