Isolation of Chlorophylls and Beta Carotene from Plant Leaves
Wang Haina A0133901R
1. Aim
1. To isolate chlorophyll and beta carotene from plant leaves using column chromatography.
2. To qualitatively characterise the pigments with UV-vis spectroscopy and TLC.
2. Results and discussion
Isolation of beta carotene and chlorophylls by column chromatography
Upon the loading of S1 (the extract of the plant leaves in hexane), a yellow band appeared at the top part of the silica column immediately after the solvent level descended to just above the sand layer. This yellow band later developed to about 2 centimetres long, and moved downwards almost as quickly as the eluent (hexane). This band was possibly beta carotene, as beta carotene is a non-polar molecule, and interacts well with the non-polar solvent, but poorly with the polar static phase (SiO2). The yellow band was collected and labelled S2. No colour band was seen above this yellow band, but in the meantime, a dark green layer was seen attached to the top of the upper sea sand layer, which meant that there were pigments with higher polarity in the extract that did not dissolve substantially in hexane.
Upon the addition of 10 mL of hexane: ethyl acetate (1:1), a green solution was immediately formed above the sand level. As this solution entered the column, three colour bands were formed. The lowest was green, and the second lowest was yellow. However, these two bands were very close together, forming a rather continuous band, as no apparent boundary was observed between them. The colour changed gradually from green to yellow.
This poor separation was probably due to the close polarity of the green and the yellow compounds, but the reason was also highly likely that I added the 10 mL of eluent directly with a beaker, instead of adding slowly with a dropper in a circular manner. As a result, on the first impact between the sand layer and the falling eluent, a small amount of air was inevitably introduced into the silica gel. Although the sand layer was later tapped to be level again, air bubbles were sealed inside the column, and moved downwards with the eluent. This explains why during this separation, many air bubbles were seen on the inner wall of the column, causing cracks from time to time, despite the fact that the column did not have bubbles or cracks until the end of the collection of S1, and that the column was never run dry. Bubbles and cracks often cause poor separation.1 Nevertheless, the green band was collected on the basis of an estimated boundary and labelled S3. Judging from the green colour, it should contain mainly chlorophylls.
Another band, which was also the highest, was yellow as well, and appeared to reside constantly at the very top of the column. This suggested that it consisted of very polar compounds that clung tightly to the static phase. The eluent was changed to 100% ethyl acetate to increase its mobility until it was collected. No colour band was observed after the collection of this band.
Characterisation of beta carotene and chlorophylls by UV-vis spectroscopy
The spectrum of S2 (Figure 1a) gives a peak at 450 nm and a lower peak at 475 nm. This is in good agreement with the literature peak wavelengths of beta carotene in hexane given by Jeffery2, i.e. 499.9 nm and 477.7 nm. Jeffery2 also points out a region where the slope is smaller than its neighbour points at around 422 nm. This feature is also seen in the spectrum of S2. Therefore, beta carotene was isolated successfully. The discrepancies between the experimental results and the literature values may be due to small amount of impurities that had similar polarity with beta carotene or different properties of spectrophotometers, as well as minor difference in the composition of the solvent, as it was a mixture of various isomers of hexane, and may differ in this experiment and Jeffery’s.
Figure 1 The experimentally obtained spectrum of (a) S2, and (b) S3.
The spectrum of S3 (Figure 1b) exhibits various peaks. The most significant ones are the highest and widest peak at 410 nm, with absorbance 0.6718, a peak at 454 nm with absorbance 0.2843, which appeared to be ‘affiliated’ to the previous one, and a rather sharp peak at 664 nm with absorbance 0.3679. The absorbance was low from 500 nm to 610 nm, and beyond 800 nm.
The pattern of two peaks at 400+ nm and 600+ nm respectively is adopted by the spectra of both chlorophyll a and chlorophyll b, as can be seen from the literature result in Figure 2.3 Therefore, it is certain that S3 contained chlorophylls.
Figure 2 Literature absorption spectra of chlorophylls a and b.3 A set of more detailed spectra with molar absorptivities at all wavelengths are given by Prahl.4 5
Now the problem remains that whether both chlorophylls a and b were present in S3, and whether there were impurities present. The literature wavelengths of absorption maxima (λmax) of chlorophyll a are 430 nm and 662 nm, andλmax of chlorophyll b are 453 nm and 642 nm. Therefore, the peak at 454 nm in the spectrum of S3 is reasoned to be contributed by chlorophyll b, and the peak at 664 nm contributed by chlorophyll a. Therefore, S3 contained both chlorophylls. The discrepancies are similarly discussed as in the case of beta carotene.
However, the ‘major’ peak at 410 nm was at a wavelength shorter than any of the individualλmax of chlorophylls a and b. This suggests that the S3 does not contain only chlorophylls a and b, because if we assume that chlorophylls a and b were the only solute in S3, then the overall absorbance at a given wavelength would be a linear combination of the individual absorbances of chlorophylls a and b at this wavelength6, the coefficients being their respective concentrations, i.e.
where A refers to the respective absorbances (at wavelength ). and are concentrations of chlorophylls a and b in S3, and and are the molar absorptivities of chlorophylls a and b. b is the length of the cuvet (1 cm). From Figure 2 it is clear that , and . Therefore, . This contradicts the experimentally obtained result that the highest peak was at 410 nm. Therefore, there must be pigments other than chlorophylls a and b which absorbed light significantly at below 430 nm. As a large absorbance in the violet region tends to make the colour of the sample more yellowish (violet and yellow are complementary colours), we may expect the impurities to come from the yellow band above the green band in the column chromatography, since the two bands were poorly separated.
TLC analysis
The TLC results of S1, S2, and S3 are shown in Figure 3.
Figure 3 TLC results of S1, S2, and S3.
The samples S1 and S3 were too concentrated that the spots were elongated into streaks. However, it can still used to confirm that S2 contained the least polar pigment, beta carotene, and S3 contained chlorophylls. The yellow spot at the bottom of S1 corresponded to the third and fourth colour bands in the column chromatography. There was also an apparent grey spot in the route of S1, which was not discovered in column chromatography. This grey pigment should have been drained out completely before the collection of S3, due to its significantly lower polarity than chlorophylls shown here by TLC. However, the path of S3 also showed a faint grey, which indicated that the separation of the grey pigment and the chlorophylls was not satisfactory due to the bubbles and cracks.
According to colours and Rf values of plant pigments summarised by University of Pittsburgh at Bradford7, the grey pigment was likely to be pheophytin a, and the yellow spot at the bottom of S1 may consisted of various xanthophylls.
3. Conclusion
In conclusion, of the two samples collected from column chromatography, S2 contained almost pure beta carotene, while S3 was confirmed to contain various impurities other than chlorophylls a and b. Among them are xanthophylls and pheophytin a. The apparent presence of impurities was mainly due to poor separation caused by bubbles introduced into the column upon the top-up of the eluent. On the other hand, as a way to improve the experimental methodology, the elution could be divided into more steps, with a different eluent used in each step, in order to facilitate good separation. Also, TLC is best to be run before the column chromatography to indicate what colour bands are to be expected.
References
1 Millar S. Tips and Tricks for the Lab: Column Troubleshooting and alternatives. http://www.chemistryviews.org/details/education/2345141/Tips_and_Tricks_for_the_Lab_Column_Troubleshooting_and_Alternatives.html (accessed October 8, 2014).
2 Jeffrey, S. W.: Beta-beta-carotene. In Phytoplankton pigments in oceanography: guidelines to modern methods; Mantoura, R., Wright, S. Eds., Bremerhaven: Pangaea, 1997.
3 Austin Community College. How can a scanning spectrophotometer be used to analyze the purified pigments that were separated using TLC? http://www.austincc.edu/biocr/1406/labm/ex7/prelab_7_4.htm (accessed October 8, 2014).
4 Prahl S. Chlorophyll a. http://omlc.org/spectra/PhotochemCAD/html/123.html (accessed October 8, 2014).
5 Prahl S. Chlorophyll b. http://omlc.org/spectra/PhotochemCAD/html/125.html (accessed Octorber 8, 2014).
6 Chemistry Experiments for Instrumental Methods, by Sawyer, Heineman, and
Beebe, John Wiley & Sons, 1984
7 Thin layer chromatography of vegetables. https://www.upb.pitt.edu/uploadedFiles/About/Sponsored_Programs/Science_In_Motion/Chemistry_Labs/chem014_Thin%20Layer%20Chromatography%20of%20Vegetables.doc (accessed October 8, 2014).