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1 Alyssa Ritter, CHEM213B Synthetic #1 FFR Experiment #23: Column Chromatography and UV/Vis of Vegetable Pigments Introduction Natural

products are extremely useful for organic chemistry, especially in the development of medicines and for the discovery of new biologically useful compounds naturally occurring in various organisms. Chemicals found in nature have been commonly used for medical treatments and up to 60% of the world relies on natural compounds as their single source of medicine.1 The extraction, purification, and determination of the function and purity of new natural chemical compounds is important for the development of new and safe pharmecueticals.1 The naturally occurring compound -carotene, a carotenoid found in carrots and other dietary sources, has been shown to be an effective antioxidant, possibly providing some protection from the development of cancer.2 -carotene is a precursor to vitamin A, which is an essential nutrient that promotes cell differentiating actions and plays a role in the immune systems functioning; therefore, -carotene is extremely important biologically as it aids in the proper functioning of the body.3 Another naturally occurring carotenoid is lycopene, most

commonly found in tomatoes, which is also a precursor to vitamin A.4 Lycopene is of even greater importance than -carotene because it has the greatest antioxidant activity of all of the dietary carotenoids4, and therefore offers greater protection against oxidation and certain cancers. Lycopene can be found in both fresh tomatoes and in tomato paste, but the body is better able to use the lycopene when it is ingested in tomato paste. While -carotene has been shown to have no significant effect as a treatment for lung cancer3, lycopene has been shown to have some protective effect against cancer, specifically against the formation of breast-tissue tumors4, as well as digestive and cervical cancers.5

2 Lycopene and -carotene are clearly very biologically important chemicals with similar structures and both are precursors to vitamin A (Figure 1). Lycopene is one of the most unsaturated compounds commonly found in nature as seen by the presence of thirteen carboncarbon double bonds and various methyl groups. -carotene is also highly unsaturated and contains two cyclic groups on either end.5 Figure 1: The structures of lycopene, -carotene, and vitamin A5

Both of these compounds are highly colored, and the difference in their color is due to the differences in their structures and the separation of their and * orbitals.6 Lycopene absorbs light with a smaller wavelength than -carotene, which corresponds with less energy needed to promote an electron from the to the * orbital. Therefore, UV/Vis analysis can be used to identify these compounds from each other. These differences in their structures also are the cause of their differing polarities; -carotene is less polar than lycopene. The purpose of this experiment was to isolate these compounds from a mixture of tomato paste and carrot puree, and then separate the two pigments using column chromatography.5 Extraction of the organic pigments from the non-organic materials also in the starting material was done by mixing the starting material with ethanol, and then refluxing the resulting solid with dichloromethane.

3 Column chromatography was used to isolate and purify the lycopene and -carotene.5 They were then identified using UV/Vis spectroscopy analysis. This experiment is important because it is investigating one method of isolation and purification of -carotene and lycopene that could possibly be used for the pharmaceutical development of these substances as supplemental antioxidants. Experimental: Isolated Compounds, Lycopene and -Carotene from Tomato Paste and Carrot Baby Food. Lycopene and -Carotene. A mixture of tomato paste and carrot puree (13 g) was dissolved with 95% ethanol (30mL) and stirred for 5 minutes. The solids were extracted using vacuum filtration with a Bchner funnel and then placed in a 25mL round bottom flask with dichloromethane (10mL). The mixture was refluxed for 5 minutes, cooled, and the solids vacuum filtered with a Bchner funnel; the extraction was repeated two more times. The dichloromethane extracts were washed with saturated sodium chloride solution (2 x 10mL). The aqueous layer was removed and the resulting dichloromethane was dried with anhydrous sodium sulfate; the solvent was decanted and evaporated with nitrogen. Residues were re-dissolved in hexanes (1mL). Thin-layer chromatography (TLC) analysis was performed (2% dichloromethane/hexanes); -carotene eluted farther (Rf value = 0.132) and then lycopene (Rf value = 0.026). The pigments were separated on an alumina column using hexanes as the initial mobile phase, 2% dichloromethane/hexanes after the sample separated, and 100% dichloromethane after the yellow band eluted. The column was flashed with nitrogen to increase the elution rate. The separation was monitored using TLC (2% dichloromethane/hexanes) and like fractions combined. The solvents were evaporated and pigments were dissolved in 10% dichloromethane/hexanes (10mL). -carotene was isolated as a

4 yellow residue (Rf value = 0.172); UV/Vis max 458nm. Lycopene was also isolated as a deep red residue (Rf value = 0.024); UV/Vis max 478nm. Results and Discussion In this experiment, the pigments lycopene and -carotene were isolated from tomato paste and carrot puree, respectively, and then separated using column chromatography, in order to assess the effectiveness of these techniques at the isolation and purification of these biologically important molecules. These pigments were extracted from the starting mixture of tomato paste and carrots using ethanol to first remove polar substances also contained in the starting materials. Then, reflux with dichloromethane extracted the organic pigments from the non-organic materials within the mixture. The resulting product was a highly colored, deep redorange liquid containing lycopene and -carotene. Both pigments absorb light in the 400-500nm range5, which means light is reflected in the 550-650nm range6, resulting in the observed redorange color. The solids from the extraction were duller in color, but not all of the pigment was extracted as they were still a muted red. However, the extraction was extremely effective because when the sample was separated via column chromatography, the column was overloaded with sample. Once the pigments were isolated, they were analyzed using thin-layer chromatography (TLC) and then separated using column chromatography. TLC of the extracted pigment revealed that two pigments were indeed extracted. A carotene standard and the extracted pigments were compared and the respective Rf values were calculated, as seen in Table 1. Multiple spots were seen for the extracted sample which confirms the presence of multiple compounds. A dark red spot remained fairly close to the starting point and a yellow spot eluted the farthest; this is in agreement with the prediction that -carotene, the yellow pigment would elute first, followed by lycopene, the red pigment. Because of the cyclic

5 functionality on either end of the -carotene molecule, this molecule is less polar than lycopene, and will therefore elute first as it has less attraction to the stationary phase than lycopene. Table 1. Rf values of the extracted pigments as compared to -carotene (see Notebook page 49 for lane identifications). Lane/Spot Distance Traveled Rf value (mobile front = 3.8cm) S 0.4 cm 0.105 P1 0.1 cm 0.026 P2 0.3 cm 0.0789 P3 0.5 cm 0.132 The yellow-spot had an Rf value of 0.132 similar to that of -carotene standard, 0.105. The spot that eluted the latest from the extracted pigments, P1, had an Rf value of 0.026 and a bright red color, which most likely was the lycopene. Column chromatography, using an alumina stationary phase, was used to separate the two pigments from each other, based on their differing polarities. In the column the pigments were mixed and dark red-orange in color; after some separation the pigments were still orange in color although one was redder and one was more yellow. The final extracted pigments were a bright red and yellow, both highly saturated in their solvents. -carotene was predicted to elute first, as in TLC, and was indeed the first pigment to elute off of the column. Once -carotene eluted, lycopene was run off the column using a more polar solvent. TLC was used to monitor the fractions as they eluted; the first five fractions were combined as they were yellow in color and TLC indicated that the last two yellow fractions were pure (Rf = 0.172), making all previous fractions pure as well. Table 2 includes the Rf values from the TLC monitoring of the column chromatography.

6 Table 2. Rf values of the fractions from column chromatography. Lane/Spot S Y1 Y2 1 2 1st spot 2 2nd spot 3 1st spot 3 2nd spot Distance Traveled 0.4 0.5 0.5 0.1cm 0.3cm 0.65 cm 0.4 cm 0.7 cm Rf value 0.138 0.172 0.172 0.024 0.073 0.159 0.098 0.171

Lane on Plate S Y1 Y2 1 2 3

Compound -carotene standard 4th yellow fraction 5th yellow fraction Last red fraction to elute 2nd red fraction to elute 1st red fraction to elute

For the red lycopene-containing fractions, only the final fraction was pure as indicated by only one spot present on the TLC plate. This spot had a very similar Rf value to the other red spot seen in the previous TLC analysis, 0.024 and 0.026 respectively. Overall, as indicated by the TLC and the pure fractions that were obtained, column chromatography was an efficient and effective technique for the separation of -carotene and lycopene. Due to overloading of the column and a slightly short column, separation was slightly difficult, but considering only 2 fractions contained a mixture of the compounds, this was still an effective technique for separation. Because of the good separation between the pigments and the short amount of time it takes to run the column, column chromatography is a good method to use in future experiments for the purification of these compounds. UV/Vis spectroscopy analysis was then performed on the yellow and red pigments to confirm their identities. As seen from the spectral data (Figures 2 & 3, Supplemental Information), -carotene had a maximum absorbance (max) of 458nm with absorbance value 0.18089AU, which is similar to its standard of around 448nm.5 This corresponds with the observed yellow color since it absorbs light in the blue wavelength range and therefore reflects yellow-orange light. Lycopene had a max of 478nm with absorbance value of 0.59283AU, which also is very similar to the standard value of around 473nm. This also corresponds to its

7 observed color, since it absorbs light in the upper range of the blue wavelengths and reflects light of the orange-red wavelength. Both of these analyses showed pure samples as -carotene had only one peak in the visible range and the other peak in the visible range for lycopene does not correspond with a -carotene impurity. Since there is some variability in how these molecules absorb light based on the solvent in which the pigments are dissolved, this data can be used to conclude that -carotene and lycopene were indeed isolated and purified successfully. Overall, the method of extraction using ethanol and reflux with dichloromethane to yield lycopene and -carotene was extremely effective at isolating these compounds from a tomato paste and carrot puree mixture. The reaction yielded high amounts of both pigments in a concentrated and highly colored solution. Column chromatography was also very successful at separating and purifying the two compounds. TLC indicated that there were many pure fractions of -carotene and one pure fraction of lycopene, which confirms the effectiveness of column chromatography at recovering the products and also at purifying the two pigments. UV/Vis spectroscopy analysis indicates that the correct pigments were isolated and based on the accurate max values, further confirming the validity and effectiveness of these techniques. This experiment can be considered for the pharmaceutical development of these two compounds, providing a good technique for the isolation and purification of these compounds that is relatively simple and results in high yields of both -carotene and lycopene. Because these two compounds have been shown to protect against oxidation and certain cancers3,4, finding new, and cheaper, ways to produce them is important; this experiment provides one procedure for the development of these compounds. The isolation and purification of lycopene and -carotene as described in this experiment are simple, yet very effective ways of producing these two

8 biologically important compounds, and these methods should be considered in future experiments regarding these compounds.

References 1. Bankova, Vassya. Natural Products Chemistry in the Third Millenium. Chemistry Central Journal. [Online] 2007, 1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1975825/ (accessed Nov 10, 2012). 2. Terao, J. Antioxidant Activity of -Carotene-Related Carotenoids in Solution. LIPIDS. [Online] 1989, 24, 659-661. http://www.ncbi.nlm.nih.gov/pubmed/2779372 (accessed Nov 8, 2012). 3. Omenn, Gilbert S.; Goodman, Gary E.; Thornquist, Mark D.; Balmes, John; Cullen, Mark R.; Glass, Andrew; Keogh, James P.; Meyskens, Frank L. Jr.; Valanis, Barbara; Williams, James H. Jr.; Barnhart, Scott; Hammar, Samuel. Effects of a Combination of Beta Carotene and Vitamin A on Lung Cancer and Cardiovascular Disease. The New England Journal of Medicine. [Online] 1996, 334,18, 1150-1155. http://www.ncbi.nlm.nih.gov/pubmed/8602180 (accessed Nov 8, 2012). 4. Grtner, Christine; Stahl, Wilhelm; Sies, Helmut. Lycopene is More Bioavailable from Tomato Paste than from Fresh Tomatoes. Am J Clin Nutr. [Online] 1997, 66, 116-122. http://www.ncbi.nlm.nih.gov/pubmed/9209178 (accessed Nov 8, 2012). 5. Rummel, Sheryl A. Experiment 23: Column Chromatography and UV/Vis of Vegetable Pigments. [Online] 2011. https://cms.psu.edu/section/default.asp?id=MRG-120814-145425SAD270 (accessed Oct 19, 2012). 6. McMurray, John. Organic Chemistry, Eigth Edition. Lockwood, Lisa, Ed.; Brooks/Cole Publishing: Belmont, CA, USA. 2012.

Supplemental Data UV/Vis Spectra from -carotene and Lycopene (Figures 2 & 3 respectively) Notebook Pages (pages 49-51)