Investigating the Techniques of Solid- Liquid Extraction by isolating lycopene from tomato paste and Column Chromatography &Thin-Layer Chromatography (TLC)by purifying lycopene Mengying Li Department of Chemical Engineering, The Pennsylvania State University, PA 16802 Abstract mfl5156@psu.edu The separation technique of solid-liquid extraction was used to isolate lycopene from tomato paste. Column chromatography was also used to purify lycopene from the other carotene compounds such as Gr beta-carotene in the crude product. TLC test was performed afterwards to show the effectiveness of purification. An UV/Vis spectrum of the purified lycopene was also performed and analyzed in the end of the lab. Introduction Lycopene [1] is a bright red C 40 carotenoid pigment found in tomatoes. The rich concentration of lycopene in tomato is proved to be helpful in bone health due to its outstanding antioxidant properties. (1) Figure 1. Structure of lycopene Lycopene can be isolated from tomato paste (baby food) through solid- liquid extraction. In solid- liquid extraction, a solvent that has distinct properties such as hydrophilic or hydrophobic is added to a solid compound. The soluble material is extracted into the solvent from the solid compound and insoluble material is left behind with the solid compound thus completes the separation. Lycopene, as an organic hydrophobic compound, will be extracted into the hydrophobic organic layer and separated from other water-soluble compounds and solid residue. Other hydrophilic substances, such as acetone, will be easily extracted into water and sodium chloride thus it purifies lycopene to some extent. Column chromatography is a great way to separate and purify compound from the impurities that have similar characteristic with the compound interested. While solid- liquid extraction can extract lycopene from tomato paste, it also extracts some impurities that are closely related in structure with lycopene. In this lab, we will use column chromatography to separate lycopene from other carotenoid compounds such as carotenes and xanthophylls. Column
chromatography separates compounds based on their polarity. In this lab, alumina will be used as the polar stationary phase and hexane as the nonpolar mobile phase. When the analytes are eluted with the mobile phase in the column, the more polar compound will be eluted slowly since it will have a stronger interaction with the stationary phase. The less polar compound will be eluted faster since it tends to stick with the nonpolar solvent due to like dissolves like principle. Different polarity and different retention time is the key of separation. Thin-layer-chromatography, or TLC, is often used to determine the effectiveness of the purification. (3) TLC has the same separation principles as the column chromatography, but it has a different set up. The stationary phase is usually a silica plate. The analytes are dipped onto the plate and mobile phase is gradually climbing upon the plate and causes the movement of the spots. The spots can be visualized by UV light or iodine chamber. The retention factor Rf can be calculated depends on distances that the spots traveled and the distance solvent travels. The best separation are usually achieved when the Rf values fall between 0.3 and 0.7 (3) Result and Discussion Solid- liquid extraction of lycopene The solid- liquid extraction was proved to be an effective method for isolating lycopene from tomato paste. Tomato paste was first extracted with 10ml acetone and then extracted 3 times with 10ml dichloromethane. The combined filtration was treated with water and sodium chloride to break the emulsions and remove acetone and other water-soluble component. (3) The water- insoluble, orange hydrocarbon carotenoid was then separated into the organic layer and dried over anhydrous calcium chloride. Dried carotenoid was dissolved in a few drops of dichloromethane and a TLC test was then performed on the mixture. The mobile phase of TLC consisted 8ml hexane and 2ml acetone. One spot was made of high concentration mixture by repeatedly touching the plate in the same location, and the other spot was made of diluted mixture by touching the plate only once. As the elution was completed, the spots were visualized by putting the plate into the iodine chamber since no spots were detected by eyes and UV lights. Align with the concentrated spot, there were two spots with Rf value of 0.418 and 0.945. Align with the diluted spot, only one spot was seen with Rf value of 0.945. It can be concluded that there existed two components in the orange carotenoid solution. The reason that there was only one spot in the diluted one could be assumed that the amount of one of the component was too little in the diluted solution thus it was hard to detect its existence. After the TLC test, the rest of the dichloromethane solution was dried over a steam of nitrogen gas and stored in the dark place. Column Chromatography Column Chromatography was also proved to be a successful method for separation. In an 8 cm chromatography column, 3095mg alumina was prepared as the solid phase and hexane was prepared as the liquid phase. The dry carotenoid was dissolved in a few drops of dichloromethane. A TLC test was first
performed on the carotenoid solution before the column chromatography. Hexane was first washed down the inside of the chromatography column in order to consolidate the carotenoid mixture at the top of the column. (4) The column was then eluted with hexane. First two tubes of liquid that coming out of the column were colorless thus were discarded. The color of following tubes of liquid were yellow orange, orange and yellow. After this, the color of the liquid eluted was colorless. TLC test was performed on the liquid that had color. For each tube, two spots were made, one was the concentrated spot and the other was diluted spot. Spots were visualized by UV light and iodine chamber. The result of the TLC plate was shown in table 1. Table 1 Rf Values of the TCL Test on column chromatography. Concentrated spot Diluted spot Carotenoid Solution 0.54 0.54 Tube #1 0.36, 0.54 0.36 Tube #2 0.843 0.843 Tube #3 0.145, 0.843 0.145, 0.843 Tube #4 0.145, 0.54 0.145 The TLC test showed that the original carotenoid solution had an Rf value of 0.54. Tube #1 mainly had one compound that had an R f values of 0.36. Tube #2 and tube #3 all had the same major component that had an Rf value of 0.843. In tube #4 the major compound had an Rf values of 0.145. The solution from tube #2 and Tube # 3 were combined since they had the same major compound. The solution from Tube #1, Tube #2&3, and Tube #4 were concentrated to solid by nitrogen gas and stored in the dark place The TLC test showed that there were more than one spots in one plate. This situation can be assumed that two or more substances might co-elute at the similar time. Putting smaller amount of the solution eluted in the tubes might help with this problem. TLC plates also showed that there were more spots in one plate after the column than before. Before the column chromatography it had been confirmed that there were only two substances in the mixture. (after solid-liquid extraction) After the column chromatography, 4 different Rf values were observed. This circumstance might cause by the different concentration of the same substance in the original mixture. The original carotenoid mixture had not been mixed and shaken well and the same substance might have different concentration in different places in the beaker. Only one Rf value was seen for the original carotenoid solution. This can be assumed that the analyte analyzed on the TLC plate might be extracted from the same place where the substance s concentration was uniformed. UV/Vis spectrum 2-3 drops of ethanol was added to each 3 vials of dried carotenoid pigment and UV/ Vis spectrum was performed on these 3 samples. The result was showed in table 2.
Table 2 Result of UV/Vis Spectrum Wavelength (nm) Vial #1 464 Vial #2 467.4 Vial #3 472.2 Since in standard solution, β -carotene has the maxima absorbance of 448nm and lycopene has the maxima absorbance of 472nm and γ carotene has the maxima absorbance of 462nm.( Tan and Soderstom, 1989) It can be assumed that in vial #1 and #2 the carotenoid are mostly γ carotene and in vial #3 the carotenoid is mostly lycopene. This further confirmed the successful separation of lycopene from other impurities. Conclusion The solid- liquid extraction was proved to be effective when isolating lycopene from tomato paste since the TLC test showed that there were only two components in the colored organic layer. Column chromatography was also a great way to separate lycopene from other carotenoid compounds that have similar characteristics. The TLC test and the UV/ Vis spectrum confirmed that major component in the solution in the tube #4 and vial #3 were lycopene. In the future, when catching the solution eluded from the column, putting smaller amount of solution into each tube might help in separation better since the TLC plates showed that there were different compounds in the same tubes. Before doing the column chromatography and the TLC test it is also important to shake and mix the solution well in order to achieve the uniform concentration for the same substance in order to avoid the situation that more spots were observed in the TLC plates than the spots observed before the column in this lab. Experimental General Methods All compounds were purchased from Sigma- Aldrich and used without further purification Lycopene (1) Tomato paste (0.5310g) was extracted with 10ml acetone first and then 10ml dichloromethane for 3 times. The organic layer was dried over anhydrous calcium chloride. The stationary phase for TLC was silica and for column chromatography was alumina.. UV (EtOH) λmax 472.2nm. Acknowledgement I would like to sincerely and profusely thank my chemistry lab TA Steven Taylor for his guidance to the instrument usage and great support in completing my project.
Reference (1) The George Matelijan Foundation. Tomatoes [online] http://www.whfoods.com/genpage.php?tname=foodspice&dbid=44 (accessed Mar 13, 2016) (2) Riley, J. S. Liquid-Solid Extraction. DSB Scientific Consulting [online] http://www.dsbscience.com/freepubs/forensic_intern/node22.html (accessed Mar 13, 2016) (3) Williamson, K,L; Masters, K, M. Macroscale and Microscale Organic Experiments, 6 th edition. Cengage learning, Inc, 2011. p165, p179 (4) Masters, K. M. Chem 213M: Food Science Module Lab Guide, Spring 2016 edition. (5) Tan, B. ; Soderstom, D. N. Qualitative aspects of UV-vis spectrophotometry of beta-carotene and lycopene B.N.J. Chem Ed, 1989, 66,258