Iisolation of Chlorophylls and Beta Carotene from Plant Leaves
iIsolation 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
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
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). Appendices Data sheets and spectra: see attached papers.