Post lab 5

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Feb 20, 2024

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POST LAB: LAB 5 SEPARATION OF SPINACH PIGMENTS BY TLC AND COLUMN CHROMATOGRAPHY CHEM 3702L SEC 001 I. Observations Part of Procedure Observations A. Spinach pigments extraction When adding the spinach paste, composed of 10g of fresh spinach and 15 mL of ethyl acetate, to the separatory funnel, the separation of the dark green organic layer and the yellow aqueous layer occurred quickly. The yellow aqueous layer was denser making it the bottom layer in the funnel. A. Spinach pigments extraction When adding 1.25 g of anhydrous sodium sulfate to the dark green organic layer, it absorbed the polar compounds acting as a fast-drying agent. C. TLC Analysis Once the TLC plate was dropped into the developing chambers, the solvent front moved quickly to the top of the plate revealing the distance traveled by the solvent. D. Column Chromatography separation After adding the silica gel, sand, and spinach pigments, the eluting process began. Adding the 30 mL pure hexane eluded a yellow band. Adding 27 mL hexane and 3 mL acetone eluted a grey band. Adding 24 mL hexane and 6 mL acetone eluted a blue- green ,a blue, and a green band in that order which completed the elution process. II. Techniques Technique Goal How it was used in lab Thin-layer chromatography (TLC) To obtain well separated spots in order to determine the optimal solvent combinations, the progress of separation of column chromatography, ad the progress of reactions. It was used in the lab in two chambers: One chamber containing a 90:10 ratio and the second chamber containing a 70:30 ratio of hexanes:acetone. This was used to determine how the pigments separate and the Rf value for each component. Column Chromatography A purification method used to separate compounds by elution solvents to determine the polarity of the components collected in test tubes It was used in the lab to collect the five pigments, carotin, pheophytin A, pheophytin B, chlorophyll A, and chlorophyll B.

III. Data analysis Distance Travel ed by solvent: 45 mm Distance traveled by topmost spot: 1 8 mm Distance traveled by 2 nd topmost spot: 12 mm Distance traveled by 3 rd topmost spot: 6 mm Rf topmost spot: 18 mm / 45 mm = 0.4 Rf 2 nd topmost spot: 12 = 0.267 mm / 45 mm Rf 3 rd topmost spot: 6 mm / 45 mm = 0.133 Distance Travel ed by solvent: 45 mm Distance traveled by topmost spot: 1 5 mm Distance traveled by 2 nd topmost spot: 10 mm Distance traveled by 3 rd topmost spot: 9 mm Rf topmost spot: 15 mm / 45 mm = 0. 333 Rf 2 nd topmost spot: 10 mm / 45 mm = 0. 222 Rf 3 rd topmost spot: 9 mm / 45 mm = 0. 2

a. How many pigments were you able to separate by column chromatography? We separated 5 pigments from column chromatography. b. Identify the pigments and label them on the picture of your fractions from column chromatography. Are they completely separated from one another, or do they come out as a mixture of other pigments? carotin, pheophytin A, pheophytin B, chlorophyll A, and chlorophyll B. c. Rank the polarity of your pigments. Pheophytin A Pheophytin B Chlorophyll B Chlorophyll A Carotin

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Least polar : carotin, pheophytin A, pheophytin B, chlorophyll A, chlorophyll B : most polar - Less polar compounds elute faster IV. Discussion and conclusion The separation of pigments by TLC using two different solvent mixtures determines the polarity of the compound. Low polarity compounds are eluted with low polarity solvents and vice versa. Our results concluded that we had a better separation of compounds in out 70:30 ratio of hexane and acetone which entails it is more polar due to acetone being a more polar solvent. The pigments are separated in column chromatography based on the relationship between the polarity of the stationary phase and the eluting power of the mobile phase. Silica gel was the polar stationary phase with acetone as the highly polar solvent. As more acetone was added the more polar pigments eluted out of the column. V. Post lab questions Increasing order of polarity: C < A < B : B will elute last being the most polar To purify 5 g of material, we will opt for column chromatography because TLC is sued to check the progress of a reaction or the progress of separation which is only suitable for rapid analysis. So Column chromatography is appropriate so that the solute mixture is applied on tip of a long packed column. The stationary phase is polar in nature so the polar compounds from the solute mixture will absorb more strongly verses the less polar solvent.

If you wanted to check in detail you could use NMR to check the purity for each fraction collected

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