Tomato Paste
Tomato paste (10g) was weighed and the experimental procedure carried out was the same procedure as spinach above.
Thin-Layer chromatography
The spinach and tomato extracts was examined on the same cellulose thin-layer plates. A mixture of methanol:acetone:water, 15:15:1 v/v was used to develop the spots. Alumina plates was also used, which was developed by using acetone:petroleum ether, 5:95 v/v. the spots was studied under both the visible and ultraviolet light.
Results
From the two different TLC plates used, the spots observed are as follows:
Alumina Cellulose
Rf Values (Alumina) Rf Values (Cellulose)
Solvent front = 5.2cm Solvent Front = 6.2cm
Spinach Spinach
β-Carotene = 4.9, 4.9 / 5.2 = 0.94cm Chlorophyll b = 5.7, 5.7 / 6.2 = 0.92cm
Chlorophyll a = 3.5, 3.5 / 5.2 = 0.67cm Chlorophyll a = 5.2, 5.2 / 6.2 = 0.84cm
Chlorophyll b = 3.2, 3.2 / 5.2 = 0.62cm Tomato
Tomato β-Carotene = 0.1, 0.1 / 6.2 = 0.016cm
β-Carotene = 4.8, 4.8 / 5.2 = 0.92cm
Discussion
Green plants contain various types of pigment in which some of the known pigments are chlorophyll and carotenoid. Chlorophyll is what gives the colour of a green plant such as spinach its green colour and it is also known to be very useful in photosynthesis of plants, carotenoid on the other hand is a type of pigment that gives a fruits such as tomato its orange colour. Chlorophyll can be divided into two types, chlorophyll a and chlorophyll b. They both have very similar structures but the main difference in structure is the side chain. The C-O and C-N bond makes both chlorophyll a and b polar and it also contains Mg bonded to nitrogen making it a very polar bond. Chlorophyll a has a methyl (CH3) in the position where chlorophyll b has an aldehyde (CHO) side chain, which makes chlorophyll b slightly more polar than chlorophyll a with the respective molecular weight of 893.5026 (C55H72MgN4O5) and 907.4862 (C55H70MgN4O6)1. Carotenoids are tetraterpenes consisting of eight isoprene units. The orange / yellow colour of carotenoid is usually masked by the green colour of chlorophyll but when the plants starts to mature, the chlorophyll decreases having more of the orange / yellow carotenoid pigments, they are also involved in photosynthesis of the plants2.
Structure of chlorophyll a and chlorophyll b
Structure showing chlorophyll a and chlorophyll b with the C-O and C-N bond and the Mg bonded to nitrogen also showing that chlorophyll b is more polar than chlorophyll a with the side chain difference 3
Structure of β-Carotene
Structure showing β-Carotene as a non-polar hydrocarbon compound 4
β-Carotene is one type of carotenoid pigment, which gives certain fruits and vegetable their orange/yellow colour. β-Carotene is a very non-polar hydrocarbon; it is also an oxidant useful in damaging free radicals in the body. β-Carotene has a molecular formula of C40H56 (536.8824).
Performing TLC on the different type of pigment in both spinach and tomato, it is clearly obvious that the separated spot will be coloured and the carotene spot will move further up the plate than the chlorophyll spot, while chlorophyll a will move further up than chlorophyll b due to the molecular weight and polarity.
Alumina TLC Plate
Different pigments will tend to move up the alumina plate at different rates due to difference in polarity between the mobile phase and the stationary phase. The stationary phase of the alumina plate is more polar than the mobile phase making the non-polar pigment, in this case β-Carotene move more quickly up the plate than the other two pigments observed in the TLC hence β-Carotene has a higher Rf value of 0.94cm for spinach and 0.92cm for tomato due to it been non-polar and having a lower molecular weight of 536.8824, chlorophyll b been the most polar pigment is more retained by the stationary phase because the stationary phase is polar hence the lower Rf value of 0.62cm obtained and also having the highest molecular weight of 907.4862, chlorophyll a on the other hand is the middle spot as it is less polar than chlorophyll b and more polar than β-Carotene having a Rf value of 0.67cm with molecular weight of 893.5026. The spinach spot observed has both the chlorophyll a and b and β-Carotene but in the tomato spot only β-Carotene was observed, this might be due to the fact the tomato puree used for the experiment contains little or no chlorophyll in it has the tomato might has been fully matured which means that the green chlorophyll pigment masking the β-Carotene might have fully decreased in the tomato puree and having more of the orange / yellow β-Carotene pigment hence the result observed, although the chlorophyll pigment might be there but it was not observed in the TLC plate.
Cellulose TLC plate
Using cellulose plate as the stationary phase makes it a reverse phase TLC. A reverse phase TLC is a phase where the mobile phase is more polar than the stationary phase as the TLC plate is coated with a non-polar compound, cellulose.
Due to the stationary phase of the plate been non-polar, therefore, the β-Carotene will be more retained by the stationary phase which will now have the lower Rf value of 0.016cm regardless of the molecular weight of the pigment being the lowest, on the other hand, chlorophyll b will move further up the plate as it will not be retained by the stationary phase hence it will have a higher Rf value of 0.92cm.
From the TLC obtained in the result section, only chlorophyll a and chlorophyll b was observed for the spinach no β-Carotene was observed and for the tomato spot only β-Carotene was observed once again, which was in this case retained by the stationary phase making the spot hardly move. The highest spot observed for the spinach is chlorophyll b as it is the most polar pigment, therefore will move the fastest and the second spot is chlorophyll a, which is slightly less polar due to the difference in the side chain will move slightly slower than chlorophyll b.
This experiment shows that both the spinach and the tomato used contains chlorophyll a, chlorophyll b and β-Carotene, although we know that there are more or different other types of pigments in plants, fruits and vegetables but only these three pigments were observed in this experiment. It can also be observed that the separation of pigments as occurred via polarity of the pigments in the cellulose plate but the alumina plate separated the spots with both the polarity and the weight of the pigments.
Conclusion
The pigments were successfully extracted from the spinach and the tomato and was identified and separated by using thin layer chromatography. The polarity and weight of the pigments were also useful in allowing the separation to occur. It can be concluded that the aim of the experiment was achieved.
Reference:
- Sandra K. Koster, Isolation of Chlorophyll and Carotenoid Pigments from Spinach - http://www.uwlax.edu/faculty/koster/Spinach.htm
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Comparison of Pigments in Natural Products and Dietary Supplements (Nutraceuticals) - http://www.cofc.edu/~kinard/153L_HONS_HonorsChemistryLaboratory/Exp9_PigmentSeparations.pdf
- http://www.succulent-plant.com/glossary/images/chlorophyll.png
- http://harvardforest.fas.harvard.edu/research/leaves/images/b_carotene_formula.jpg