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My aim is to extract the various photosynthetic pigments from a grass tissue and later to separate them and determine which one is which by using thin layer chromatography.

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Klaudia Stadnicka Due 28.June.2002 Biology; IB5 LAB REPORT #7 CHROMOTEMOGRAPHIC SEPERATION OF PLANT PIGMENTS AIM: My aim is to extract the various photosynthetic pigments from a grass tissue and later to separate them and determine which one is which by using thin layer chromatography. To calculate Rf values for all of the pigments. Hypothesis: I expect to observe various pigments extracted. There will be a distinction between them and separation will occur due to the pigments' different solubility and attraction to the static phase. Prediction: I predict that separation of each of the pigments will occur and will be able to be observed. I also predict that the pigment that will travel the closest is chlorophyll b, and then chlorophyll a, followed by xantophyll and at the end there will be cartenoid pigments. Variables: There are no variables in this specific lab, because it is an observation lab, where we are meant to observe separation of pigments. METHOD: Equipment: * A few grass leaves * 2 glass microscope slides * blu tac * 1 glass micro pipette * 10 cm3 propanone (acetone) * 1 small watch glass * 1 electric hair drier * 1 very fine paintbrush * 2 - 3 TLC chromatography strips (1.25 cm x 6.7 cm) * 1 glass specimen tube, (2 cm x 7.5 cm) * 1 cork to fit the tube, with a horizontal V-slit * 7 cm3 chromatography running solvent per tube * 1 pencil * seeker (mounted needle) ...read more.


An Rf value is measured in order to represent the distance a pigment traveled in comparison to the solvent, which is also a representation of solubility of a specific pigment and their attraction to the static phase. Rf = distance run by pigments / distance run by solvent The Rf value from my experiment: Pigment Rf value Color Chlorophyll b 0.7 Dark green Chlorophyll a 0.5 Pale green Xantophyll 0.93 Yellow Though, the other group got some very nice results, where a separation could be seen very easily. We achieved what we expected a lucid distinction between the pigments, where they moved different distances depending on which pigment we were looking. The common, stated, official Rf values: Pigment Rf value Color Chlorophyll b 0.47 Dark blue-green Chlorophyll a 0.65 Green Xantophyll 0.71 Yellow-brown Phaeophytin 0.86 Yellow-green Carotene 0.96 Yellow DATA ANALYSIS: Chromatography has as its objective separation of molecules depending on their solubility (in this case it is the separation of various pigments). In paper chromatography, when you place a colored chemical sample on a filter paper, you can get the colors to separate from the sample by placing one end of the paper in a solvent. As the solvent diffuses up the paper, it dissolves the various molecules in the sample varying on the polarities of the molecules and the solvent. If the sample contains more than one color - it has more than one kind of molecule. The differences in the chemical structures of each kind of molecule, determine also their polarity, which will also alter while the structure alters. ...read more.


I suppose it might be due to the solvent's polarity. Let's talk about the second results, though, where our hypothesis is supported, because we see the same pattern as expected and the separation of pigments occurred. The Rf values are alike the ones received by the specialists. The pigments move the further, the more soluble they are, the lighter they are and the less attracted to the paper they are. There of course were some weaknesses and a few modifications might have been done. Weaknesses: Our main weakness is that we didn't make any replicates, which would make our results more accurate and reliable. We should have done a few more paper tests using exactly the same method. I feel as another weakness was that we didn't include any variables; we didn't even check how pigments of another plant would react. Modifications: The most important modification is to do replicates of the same experiment and at the end look on the average results. We could have also experimented with different stationary and mobile phases e.g. aluminum foil and spray aluminum oxide. We could have also looked on the experiment when using various solvents, which would highly affect the results. We could have also left the paper for a longer/shorter time to observe the differences. We should have ensured ourselves that the pigments were above the solvent, what is crucial, otherwise the pigments would be solved in the solvent. Maybe that is what happened in our first experiment. We could have also tested different plants and check the differences - compare the results. Sources: Some internet sites: http://www.grossmont.net/cmilgrim/Bio120/Lab/Paper_Chromotography_Results.htm http://www.mrs.umn.edu/~goochv/CellBio/labs/photosynthesislab/photo.html http://www-saps.plantsci.cam.ac.uk/worksheets/project3.htm http://www.nasaexplores.com/lessons/01-037/9-12_2.html Biology book: "Advanced Biology for You" by Gareth Williams Class notes Own knowledge ...read more.

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