Pre-Lab Report: Chromatographic Separation of Dyes and Spinach Pigments

Pre-Lab Report: Organic Chemistry 1, Lab 1: Separation of Dyes & Spinach Pigments by Column and Thin Layer Chromatography ● Introduction( Experiment 1) In this experiment, we will be conducting Column Chromatography. Chromatography, in its own terms, is a process in which it separates mixtures of two or more compounds based on their phases: Stationary phase and Mobile phase. In modern science, we determine these phases by polarity. In hindsight, these polarity phases will be determined by the structure and bonding of different molecules. In this experiment our given polar stationary phase will be silica gel (SiO2*H2O), to determine the polarity of different compounds. Compounds with higher polarity will be attracted to the gel, while nonpolar compounds will not. To determine which compounds are stationary and mobile, the compounds will be loaded with the gel and absorb it, which will dissolve into a known mobile phase, Methanol. When compounds are dissolving in the mobile phase, we can calculate the retention time of the compounds in the columns. The less time bounding to the stationary phase, the more time in the mobile phase, which will decrease the retention time. If X compound spends more time in the stationary phase than the other, it will have a longer retention time and come out of the column last. In this case, that compound is said to be more polar than the second compound. In order to shorten the retention time of compound X, increasing the polarity of the mobile phase, in which it would be methanol to water, will better compete with the stationary phase. Even though the attraction between the stationary phase hasn’t changed, the retention time has. With this knowledge, we will be separating mixtures of two different dyes provided with us, methyl orange and methylene blue. During this first experiment, we are going to develop a column chromatography that will successfully separate the two methyls at a reasonable amount of solvent, which can determine the specific amount of time it will take for it to separate. ● Procedure For Experiment 1 1: The first step is to set up 5 pipette columns 2: Place 2-4 drops of methylene blue-methyl orange mixture in the silica gel 3: Add small amounts of sand to prevent the disturbing of the stationary phase while we are adding the solvent 4: Eluate each column with any solvent of your choice, note these solvent in your data. 5: Collect the column elute in test tubes. When finish, there would be a series of test tubes with the methylene blue on top and methyl orange separated 6: Repeat until the goal of the experiment is met 7: Construct a table of the results and observations 8: Safety: wear gloves and goggles at all times! Methyl and silica gel are toxic ● Equations/Calculations for reactions in experiment 1 Equilibrium constant for chromatography: Partition coefficient (Kc) Kc= [ solute on stationary phase] / [ solute in mobile phase] Kc= [A]S / [A]M ● Trial # Solvent used Dielectric constant Observation ● Introduction( Experiment 2) For our second experiment for this lab, we will be conducting a Thin-Layer chromatography. In this experiment, it has a similar concept as to the column chromatography. The objective of this experiment is slightly different than the previous experiment. During the column chromatography, our stationary phase and the retention time was observed by how much time it took to flow through the column. In this experiment, our stationary phase is bound as a thin layer to support either aluminium or glass. Since the amount of adsorbent is less that our previous experiment, the compound separation will be less as well. Another point in this experiment is that our mobile phase will now be shown at the bottom of the TLC plate, and move in an upward direction. Organic chemists use TLC to analyze and determine the progression of a reaction from the reactant spot (X) from a reaction mixture (Y). Organic chemists use a formula, Rf, to determine these progression. If the reactant spots have different Rf values, they would assume that a new product has been formed. However, with all chemical related labs, there is always an irregularity within chemical reactions like these. SInce TLC often produces these irregularities, chemists have a hard time differentiating if the two spots are either similar or different. To prevent this irregularity from happening, they would use a co-spot (Z), between X and Y. If the co-spot separates the two spots, chemists can conclude that they are different. With this knowledge, we will be able to perform TLC and see different compounds within a simple item: Spinach leaves. Within this experiment, we will gain a complete separation of different compounds within the spinach leaves: chlorophyll a, chlorophyll b, pheophytin a, carotenes, and xanthophylls. During this experiment, we will notice different mobile phases. With this, we will be able to understand which one is the most efficient mobile phase that can cause the separation and visualization of compounds ● Procedure For Experiment 2 1. Spinach leaves will be grinded down in a mortar until they will be visibly broken down equally and homogeneously. 2. Pour in 5 mL of methylene chloride to the mixture and mix gently. 3. Use a vacuum filtration to filter the spinach mixture . 4. Pour that liquid into a funnel to test it even further 5. Add 5 mL of water to the solution; add the funnel together with the collected liquid ; mix everything together gently/. 6. Drain the organic layer from the separatory funnel into a new, clean flask and the remaining substances will be discarded. The organic layer will be also transferred back to the separation funnel. 7. Repeat steps 5 and 6 where the organic layer will again be washed with 2 X 5 mL of water. 8. The organic layer will be transferred to a 25-50 mL of Erlenmeyer flask. 9. By slowly pouring add small doses of Na2SO4 into the solution until no clumping is observed. To obtain accurate results, the solution has to be dried efficiently. 10: Put the solution into a clean test tube 11: Solution will be concentrated by evaporating around 50% of methylene chloride; when it's concentrated enough, the solution will appear between a bright- dark green. ● Procedure for TLC: 1: Obtain a TLC plate. A dot will be placed about 1cm away from the bottom up. The mobile phase must take place and be below the dot drawn. Be sure not to scrape the sicilia gel off of the aluminum backing 2: Immerse and open the end of the melting point capillary in the extract. We must observe the solution entering the capillary. If it is not observed, the end must be cut open to finally allow the flow into the open capillary tube. 3: With quick motion, apply the extract to the TLC plate on the spot drawn and allow the solvent to evaporate from the plate. Repeat until the original spot is sufficiently concentrated, a green color should be visible. The smaller the spot size applied each time, the better the separation 4:The thin-layer chromatography plate will be placed in a developing chamber and we also need to cover the beaker with an appropriate watch glass to help prevent the mobile phase from changing composition 5:When the eluent reaches 0.5-1 cm from the top of the plate, the thin-layer chromatography plate will be removed at this specific step before the solvent reaches the top. Using a pencil mark how far the solvent traveled and use this information after the solvent evaporated. 6: Repeat step 4 for each of the mobile phase compositions in the table below—you will have to prepare these yourself. ( Do not prepare more than 10 mL of each mobile phase) 7: Now decide which two TLC plates provided the best separation. If complete separation of all five pigments—possibly six if pheophytin b is obtained—was not observed, prepare a mobile phase with a polarity in between the mobile phases used for the two best TLC 8: Repeat step 7 until all 5 pigments have been observed ● Equations/ calculations related for reactions in experiment 2 Mobile Phase Concentrati on 0:100 20:80 40:60 60:40 80:20 Rf Values Retention Factor (Rf)= Rf= Dx/Ds distance traveled by solute/ distance traveled by solvent 100:0 ● Pre-Lab Questions: 1: Re-draw the molecules in your notebook and circle the more polar compound in each pair of molecules. Hint: You may want to use the eluotropic series found in your textbook, on p. 182: 2: What general conclusion can you draw about heteroatoms such as N, O, and Cl, and polarity? - Compounds containing N, O, and Cl are already considered as polar. With this polarity, the electronegativity of a compound increases. The higher the electronegativity, the higher the polarity. Since these atoms increase the polarity, we can also conclude that the higher the atoms, ( N, O, and Cl) are present, the higher the polarity of the compound 3. Which compound in each pair do you expect to elute first out of a column with a polar stationary phase? Compound 2 Compound 4 Compound 5 Compound 8 4 . Which compound in each pair do you expect to have the higher Rf value on a TLC plate? Rf value= distance traveled by solute/ distance traveled by solvent Stationary TLC plates are polar, therefore must have small Rf values. Therefore, compounds that produce larger Rf values will be: Compound 2 Compound 4 Compound 5 Compound 8 SInce nonpolar molecules travel down faster, they will have a higher Rf value when we you divide the two distances