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Separation of Chlorophyll a, Chlorophyll B, and Beta Carotene by Paper Chromatography

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    Abstract Pigments extracted from different greens have different polarities and may be different colors. Mixed pigments can be separated using chromatography paper. Chromatography paper is able to separate mixed pigments due to their polarity and solubility. Pigments of chlorophyll a, chlorophyll b and beta carotene will be separated on chromatography paper because each has its own polarity and solubility, which results in different distance traveled up the paper. Beta carotene is non-polar so it travels the highest distance, followed by chlorophyll a.

    Chlorophyll b is the most polar; therefore, it travels the shortest distance. The separated pigments on the chromatography paper can be eluted in acetone and absorbance spectrum is determined using spectrophotometer. Spectrophotometer produces a graph of the absorbance spectrum which shows the wavelengths (in nm) that are absorbed by the pigment and that are reflected. The reflected wavelength of chlorophylls a and b reflect the wavelengths of l light that are associated with green, which gives them their color.

    Beta carotene reflects the wavelengths that are associated with orange color, and that explains its color. Introduction Chromatography paper can be used to separate mixed chemicals, including mixed chloroplast pigments prepared from extract from fresh green grass or spinach. The mixture of pigments was prepared from organic greens, which were cleaned, cut into small pieces and bathed in acetone overnight (S. W. Jeffrey). The different pigments, chlorophyll a, chlorophyll b, and beta carotene have different polarities, due to which the separation of these pigments is possible with chromatography paper.

    Beta carotene is non-polar, chlorophyll b is the most polar, chlorophyll a is more polar than beta carotene, but less polar than chlorophyll b. Chlorophyll a has a methyl group in its structure where chlorophyll b has an aldehyde; this difference results in different polarities (Lampman). Chromatography paper has a stationary phase and a mobile phase. The mobile phase travels through the stationary phase and carries the pigments from the mixture with it (Paper Chromatograhy). Different polarities of the pigments play a role in their solubility.

    Chlorophyll b is the most polar pigment and it will be the least soluble in the solvent. Chlorophyll a is less polar, so it will have a middle solubility. Carotene is the least polar pigment so it will be the most soluble. Due to their solubility, The carotene will travel up the farthest on the chromatography paper, chlorophyll a will be in the middle and chlorophyll b will go up the shortest distance. The separated pigments have their own wavelength which gives them their color from the absorption spectrum. Absorption spectrum can be determined using spectrophotometer.

    The spectrophotometer produces a graph, which tells what colors are being absorbed and which are reflected(Infrared Spectroscopy in Astronomy). Chlorophylls a and b will absorb all colors but green, which they will reflect. Beta carotene will absorb all colors but orange, because the reflected color is the color of the pigment. Materials and methods To separate the leaf pigments from the extract chromatography paper was used. On the chromatography paper a pencil line was drawn 1 cm from the edge of the paper cylinder.

    Leaf pigment extract was added to the paper along the pencil line. This step was repeated 3 times and after each time the extract was set to air-dry before the step repetition. In a well ventilated area to decrease chances of inhalation, using forceps, the loaded paper cylinder was put into the solvent of petroleum ether and acetone in a jar with a lid. The lid was closed and saturated atmosphere was created. After the pigments separated on the chromatography paper, the cylinder was removed from the jar and was air-dried.

    The paper had the separated pigments and was used to analyze the data (Carter, Morgan 140). To determine what gave the pigment its color a spectrophotometer was used. The pigments extracted from the mixture by chromatography paper were eluted in beakers with acetone and the solutions were put into cuvettes. The Spectrophotometer measured the wavelength of the solution in the cuvette, producing a graph of the absorption spectrum for leaf pigments (Carter, Morgan 143). Results Chromatography paper was used to separate mixed pigments and determine their polarity.

    The spectrophotometer was used to determine the absorbance spectrum for each of the pigments. Beta carotene Chlorophyll b Chlorophyll a Figure 1 below shows the absorbance spectrum for each of the pigments extracted from the extract from fresh greens. The chlorophyll a and chlorophyll b are green in color and the spectrum shows that they absorb violet- blue and red colors, but reflect green. Beta carotene is orange, and its graph created by spectrophotometer shows that it mostly absorbs blue and green light and reflects orange.

    Figure 1. The absorption spectrum for leaf pigment, wavelength in nm. Discussion During the separation of the pigments by chromatography paper, chlorophyll b traveled the shortest distance, chlorophyll a went above it, and the highest went beta carotene. This supports my hypothesis and the predictions made prior to the experiment matched the reality. From this information the following can be inferred: the less polar pigment is, the more soluble in the solvent it is, and the more polar a pigment is, the less soluble it is in the solvent.

    Using spectrophotometer to determine the absorbance spectrum of the pigments that were separated by chromatography paper, the information about which wavelengths are absorbed and which are reflected was produced. The peaks on the graph in Figure 1 show the wavelengths that the pigments absorbed, and the places on the graph where the graph is flat represent that those wavelengths are being reflected. Chlorophyll a is reflecting the wavelengths that are associated with green color, and that gives them their color.

    Chlorophyll b has a flat point on the graph in the wavelength that is associated with green color, which means it is absorbing all colors but green. Beta carotene is absorbing all colors but orange, and the peaks at the violet-blue and green show that those colors are being transmitted. These results support my hypothesis that the colors of the pigments are the ones that are not being transmitted, but are reflected. This data shows that for everything around me the color of objects is, is actually the color of light that cannot be absorbed and therefore is reflected.

    Although the results produced by the spectrophotometer make sense to me, the experiment was not replicated, but was don’t only once. A similar experiment I have done in the past was done twice, and the graph of the absorbance slightly differed, which tells me that there may be a small percentage of inaccuracy in the results of this experiment. Also, the cuvettes used were not brand new, so they may have been slightly scratched changing the absorption spectrum due to the clarity of the cuvette. To take this experiment to the next step, it should be replicated and the data should be averaged.

    Literature cited “Infrared Spectroscopy in Astronomy. ” Infrared Spectroscopy in Astronomy. Web. 31 May 2012. <http://www. ipac. caltech. edu/outreach/Edu/Spectra/spec. html>. K. M. Davies (ed. ), Annual Plant Reviews, vol. 14: Plant Pigments and Their Manipulation [An introduction to plant pigments in biology and commerce, pp. 1–22; Chlorophylls, pp. 23–56; Carotenoids, pp. 57–91; Flavonoids, pp. 92–149], Blackwell/CRC Press, 2004 Morgan G. J. , Carter B. E. Investigating Biology: Laboratory Manual 7th Edition. Emory University and Oxford College of Emory University, Pearson Education inc, 2011. “Paper Chromatography. ” Paper Chromatography. Web. 31 May 2012. <http://www. chemguide. co. uk/analysis/chromatography/paper. html>. Pavia, D. L. , Lampman, G. M. , Kriz, G. S. , and Engel, R. G. Introduction to Organic Laboratory Techniques: A Microscale Approach 3rd Edition. Saunders College Publishing: New York, NY, l999. S. W. Jeffrey. Paper-Chromatographic Separation of Chlorophylls and Carotenoids from Marine Algae. C. S. I. R. O. Marine Laboratory, Cronulla, Sydney, Australia, 1961.

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