Paper Chromatography Lab

Purpose: to separate the various photosynthetic pigments of Spinacia oleracea leaves. Materials:

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– methanol
– water
– filter paper
– sand
– test tube
– 10 S. oleracea leaves
– 2 wooden splints
– mortar
– pestle

Method: First, 10 S. oleracea leaves were gathered. A piece of filter paper, a wooden splint, a mortar and pestle were also gathered. The leaves were placed inside the mortar, filling the mortar approximately half full. The leaves were then crushed with the pestle. Sand, water and methanol were added and then the leaves were crushed again. This was repeated until the leaves had reached the consistency of a soupy green paste. The leaf extract (soupy green paste) was strained, removing the fibrous spinach paste, which was placed into the garbage, and leaving the liquid leaf extract. The wooden splint was dipped into the leaf extract and then traced in a thin straight line onto the filter paper. The filter paper was allowed to dry for approximately 2 minutes. The next coat could not be applied until the paper was completely dry. The process was repeated 10 times, from dip, trace and drying. A test tube was gathered as well as methanol and a second wooden splint. The wooden splint was pierced through the top of the filter paper and then the filter paper was placed into the empty test tube. It was measured how much methanol would be needed so that the tip of the filter
paper was submerged but the extract line was not submerged. After measurement, the filter paper was removed from the test tube. The methanol was poured into the test tube up to the measured fill point. The filter paper was then carefully inserted so that it never touched the edges of the test tube. The test tube was carefully placed vertically into a test tube holder and allowed to absorb methanol for 45 minutes. After the 45 minutes were complete, the filter paper was then removed, measured as to how far pigments had travelled and left to dry.


Calculations: Rf xanthophyll= distance pigment travelled
distance to solvent front
= 7
= 0.72
Rf chlorophyll a= distance pigment travelled
distance to solvent front
= 8.4
= 0.87
Rf chlorophyll b= distance pigment travelled
distance to solvent front
= 8.5
= 0.88
Rf carotene= distance pigment travelled
distance to solvent front
= 8.7
= 0.90
Discussion: 1. The Mortar and Pestle is used to break down the intramolecular bonds within the S. oleracea leaves. This helped break the molecule down into smaller molecules and individual atoms so that chloroplasts and their pigments were not buried inside the leaf and were easily accessible.

2. Methanol is used to dissolve the pigments, as methanol is polar and so are the pigments. As we saw, the pigments stayed with the methanol in the liquid part of the mixture but everything else (cell walls, etc) floated to the top of the mixture, so we could easily remove it.

3. It is important that the filter paper did not touch the walls of the test tube because if touching the side walls, water would absorb from the sides as well as the bottom of the filter paper. If this were to happen, the flow of methanol up the filter paper would be disturbed, thus disturbing the flow and separation of the pigments.

4. It is very important that the leaf extract not be dissolved in the solvent because it would not travel upward the same. If the extract line were to be dissolved in the methanol, it would simply dissolve into the methanol, without actually having the chance to travel up the filter paper. With the extract line above the meniscus, the pigments’ only choice was to follow the path of the methanol.

5. Water would not be able to carry the pigments up the filter paper because it does not have the same physical properties as methanol. One of methanol’s unique features is that it dissolves compounds of very high polarity. While water will dissolve polar molecules, it would be unable to dissolve these pigments as well as methanol can.

6. According to my findings, chloroplast a is in the highest abundance in the leaf extract. This is known because of the very bright green colour of the pigment, as well as the expanse of chloroplast a on the filter paper.

7. Pigments separate rather than travel up the paper at the same rate due to their solubility. Pigments that are the most soluble in methanol will travel to the highest levels, almost as high as methanol itself, but pigments that are least soluble in methanol will stay lower down on the filter paper. The pigments will only travel far enough so that they are completely dissolved from the methanol to the filter paper. Therefore, those pigments that are least soluble in methanol, take less time and space to dissolve from methanol so they stop travelling at an earlier stage that those that are most soluble with methanol.

8. The meaning of the Rf value is to help identify pigments. Each pigment holds an Rf value, which is used like a fingerprint. Although colour can be used to identify pigments, their Rf value is the true indication of which pigment is present. Conclusion: the various photosynthetic pigments in S. oleracea were separated using the chromatography technique.

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