Lab 7: Plant Physiology (Paper Chromatography)

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INTRODUCTION

As we know, plant carried out photosynthesis and they used two pigments which can be categorize by primary pigments and accessory pigments. A pigment is a molecule that absorbs light. The primary pigments are the chlorophyll, which are called as chlorophyll a and chlorophyll b. The chlorophyll is the green pigments that absorb lights strongly in the blue and red but poorly in the green portions because of electromagnetic spectrum. Hence, the green portion of the solar spectrum is actually reflected, not absorbed.
Plant pigment can be identify through separation and isolation of the pigments using a thin layer of paper chromatography. Chromatography is used to separate mixtures of substances into their components. All forms of chromatography work on the same principle.
They all have a stationary phase (a solid, or a liquid supported on a solid) and a mobile phase (a liquid or a gas). The mobile phase flows through the stationary phase and carries the components of the mixture with it. Different components travel at different rates. In paper chromatography, the stationary phase is a very uniform absorbent paper. The mobile phase is a suitable liquid solvent or mixture of solvents.
 Through this experiment, we will see the color of plant pigment by separating it from plant by using paper chromatography and hydrophobic solvent. We may pick any of the leaves exist to see the pigments available.



OBJECTIVES
  • ·         to learn how to separate compound into their components
  • ·         to calculate the Rf value of pigments
  • ·         to identify the colour of pigments contained in a compound


MATERIALS
  • ·         Chromatography paper
  • ·         Scissor
  • ·         Plant leaves/flower
  • ·         Test tube
  • ·         Test tube rack
  • ·         Hair dryer
  • ·         Mortar and pestle
  • ·         Solvent
  • ·         Pin
  • ·         Cork



METHOD

1. Pin head was used as the dropper, chloroplast extract was drop on the prepared chromatography paper.
2. The extract was drop at 1.0 cm from the pointing end of the paper. The drop was dry with the hair dryer and the process was repeated for 3-4 times until one small dot of thick pigment available.
3. The paper strip was attached to the cork stopper by using a pin. The strip then placed vertically into the test tube that contained a solvent.
4. The solvent than let to move and the paper was removed before the solvent front reaches the top of the chromatography paper.
5. The last range of the solvent was marked with pencil.



RESULTS


1. Identify each pigment obtained from the experiment and record your result in the table.
2. Calculate the Rf value of each pigment observed.
Rf =  Distance travelled by compound/ distance travelled by solvent



Leaf A (Red spinach)
No.
Color
Pigment
Rf value
1.
Yellow-orange
Carotene
8.2 / 10.3 = 0.79
2.
Yellow
Xanthophyll
5.6 / 10.3 = 0.54
3.
Blue green
Chlorophyll a
 3.4 / 10.3 = 0.33
4.
Yellow green
Chlorophyll b
 2.2 / 10.3 = 0.21

Leaf B (Green spinach)
No.
Color
Pigment
Rf value
1.
Yellow-orange
Carotene
8.7 / 10.0 = 0.87
2.
Yellow
Xanthophyll
 6.4 / 10.0 = 0.64
3.
Blue green
Chlorophyll a
 4.5 / 10.0= 0.45
4.
Yellow green
Chlorophyll b
 4.0 / 10.0 = 0.40

Question:

1. Why the developing solvent mixture is prepared fresh before use?
The developing liquid phase comprises of a pure solvent but more often it is a mixture of two or more solvents in specified proportions. In case solvents are mixed and stored for long periods there could be loss of volatile component which will alter the mixing proportions.

2. Why is it important to keep the dye spots (leaf extract) above the solvent level?
Because if the dye spots submerge in the solvent, the spot would dissolve into the solvent, this will prevent the dye from separating out and no result will obtain from the experiment.

3. Why is it necessary to cover the test tube during the paper development?
This is essential as the environment inside the test tube should remain saturated with the solvent vapour. Development times can vary from about an hour to several hours and a saturated environment prevents losses due to evaporation.

4. Why it is important to stop the chromatogram before the solvent front reaches the top of your chromatography paper?
It is important because you cannot determine the Rf value if you do so. It is important to see where it stop even if you let it go as high as you want.

5. Why is it important to mark the solvent level on the chromatography paper when you remove it from the test tube?
So that we will able to measure the solvent travelled through out the experiment. This it to get the Rf value.

6. Which of the pigments migrated the farthest and why the separation of pigments occur as it did? How does paper chromatography work? 7. Explain what would happen to your chromatogram if you let it run too long?
Carotene pigment travelled the farthest because this pigment has high solvent solubility. Paper chromatography work to separate the mixture of substance into their compound.



DISCUSSION

The number of bands appear on the chromatography paper tells us how many compound available in a substance. The distance travelled by its compound depends on the solubility of substance in the liquid solvent that was used, in this experiment we used hydrophobic solvent.For a given substance, the distance it moves depends on the total time the experiment is carried out as well as on the physical and chemical properties of the system.  
 In this experiment we choose spinach vegetables as our plant leave. Based from the result we obtained, we can see that carotene pigment  which in yellow-orange color travelled far away and fastest along the chromatography paper, has the highest solubility in the solvent. To be compared, chlorophyll b pigment travelled the slowest along the chromatography paper. This means that it has the lowest solubility in the solvent compared to other pigment. Carotene pigment shown to be the fastest to travelled along the paper, while chlorophyll b travel the slowest along the chromatography paper.
Hence, we learn that the choice of the solvent in paper chromatography is important as the solvent must dissolve the various components in the mixture and there must be at least slight differences in solubility of each component. If so, there will be a separation of compounds. We can see from our result that some components are less soluble, so they remain from the origin, not moving along the paper chromatography.



CONCLUSION

Based from this experiment, we can conclude that plant leaves contain of pigments called chlorophyll a, chlorophyll b, carotene and xantophyll.



 REFERENCE

Alexis Writing. (2014, September 20). How Does Paper Chromatography Work, and Why Do Pigments Separate at Different Points?. Retrieved from https://sciencing.com/how-does-paper-chromatography-work-and-why-do-pigments-separate-at-different-points-12750741.html

Schoolworkhelper Editorial Team. ( June 2017). Chromatography lab answer. Retrieved from https://schoolworkhelper.net/chromatography-lab-answers/





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