Lab report 5 PDF

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Summary

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Objective: The objective of this lab was to extract chlorophyll and carotene pigments from spinach. ThinLayer (TLC) Chromatography was used to reveal the number of components in the mixture. Wet Column Chromatography was used to separate and isolate the fractions. The goal of this experiment was to conduct TLC on a sample mixture and to analyze the eluted fraction to determine the presence of products.

Procedure: Thin-Layer Chromatography (TLC) Procedure 1. Approximately 0.5g of spinach leaves were weighed out. The leaves were torn into little pieces and placed into a mortar. Approximately 1.0mL of acetone and hexanes along with sand were added. The mixture was grinded until the liquid turned a bright, deep green. 2. The liquid was transferred to a centrifuge tube using a 5” Pasteur pipet. The mortar was rinsed with approximately 1.0mL of acetone and all the content was transferred to the same centrifuge tube. 3. Approximately 1.0mL of hexanes and 2.0mL of distilled water was added to the extract in the centrifuge tube. A Pasteur pipet was used to gently draw the mixture in and out the tube until the two layers had been thoroughly mixed. 4. A 9” Pasteur pipet was used to draw off the aqueous layer and transferred to a small beaker. Approximately 2.0mL of distilled water was added to the centrifuge tube as a wash. The aqueous layer was then drawled off and combined with the first one in the beaker. 5. The organic extract was dried over Na2SO4 in the centrifuge tube. 6. To remove the drying agent and to filter the extract, a filter pipet was prepared by inserting a small cotton plug into a 5” Pasteur pipet. The pipet was secured on the ring stand and a vial was placed below the pipet. The transfer was completed by rinsing the centrifuge tube containing the drying agent with another 1.0mL of hexanes. 7. A TLC plate was obtained, and it was spotted with the extract. The plate was developed in a solution of 7:3 hexanes: acetone. A paper towel was placed in the chamber and a watch glass was placed over it. 8. A UV lamp was used to ensure that all possible spots were noted. 9. Rf values were determined for all the spots.

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Observations 0.521g Acetone was clear, had a strong smell, and drying on the skin Hexane was clear The liquid looked like spinach soup and smelled like freshly cut grass

This process was tricky The bottom layer was cloudy and light green The top layer was clear and bright green

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The aqueous layer was a nearly colorless with a tint of green to it

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The Na2SO4 was clear and powdery It looked like salt/sugar The extract flowed smoothly through the filter pipet

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6 spots appeared Yellow, light green, and dark green

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One gray spot appeared under the UV lamp .09-xanthophyll

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.20-xanthophyll .31-xanthophyll .32-chlorophyll B .41-chlorophyll A 0.49-pheophytin B 0.86-carotene

Column Chromatography Procedure 1. A microcolumn was obtained and a cotton plug was inserted. The column was clamped on a ring stand and a beaker was placed beneath it to collect the excess solvent. 2. Approximately 4.0g of silica gel was weighed out into a small beaker and hexanes were added. It was stirred until a slurry was created. 3. The slurry was pipetted into the column using a 5” Pasteur pipet. Some of the hexanes were occasionally pipetted into the column to maintain the liquid level. A small amount of sand was added to the top of the column. 4. A 5” Pasteur pipet was used to load the sample on to the prepared column. Once the extract travelled into the sand layer, approximately 0.5mL of hexanes were added to the centrifuge tube. 5. Hexanes were used to begin eluting. Once the yellow band was approximately ¾ of the way down the column, fractions were collected in test tubes and vials. 6. Ethyl acetate was added to the eluent to increase the polarity of the solvent. Fractions were collected until the final colored band was eluted. 7. A larger TLC plate was obtained and numbered along the baseline with the number of fractions collected. The fraction was spotted multiple times. 8. The plate was developed. Rf values were collected and identifications were made.

Observations

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4.18g of silica was weighed out The slurry had a good consistency: not too thick or thin Slurry had a greyish tint Looked like gray gorilla snot

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The yellow band formed immediately Moved down the column rapidly

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A deep green ban was created Other colors like gray and yellows appeared after

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0.89-carotenes 0.65-pheophytin B 0.42-chlorophyll A 0.30-chlorophyll B 0.19-xanthophyll

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Sketches:

Data: TLC after extraction Eluent: 7:3 hexanes: acetone by volume The solvent moved a total of 5.9cm. Spot number on picture

Spot Color

Distance Spot Moved (cm)

Rf Value

Identification

1. 2. 3. 4. 5. 6. 7.

Yellow Yellow Yellow Light green Dark green Gray Yellow

0.58 1.2 1.8 1.9 2.4 2.9 5.1

Calculations: Rf =

0.58 5.9

= 0.09

TLC after column: Eluent: 7:3 hexanes: acetone by volume Fraction 1: The solvent moved a total of 4.6cm.

0.09 0.20 0.31 0.32 0.41 0.49 0.86

Xanthophyll Xanthophyll Xanthophyll Chlorophyll B Chlorophyll A Pheophytin B Carotene

lue

Identification Carotene

Fraction 2: The solvent moved a total of 4.6cm. Spot of number on picture 1. 2.

Spot color Gray Dark green

Distance spot moved (cm) 3.01 1.93

Rf value

Identification

0.65 0.42

Pheophytin B Chlorophyll A

Rf value

Identification

0.30 0.19

Chlorophyll B Xanthophyll

Fraction 3: The solvent moved a total of 4.6cm. Spot of number on picture 1. 2.

Spot color Light green Yellow

Distance spot moved (cm) 1.40 0.88

Discussion: Thin-Layer Chromatography (TLC) was a tool utilized for characterization. It assessed the purity of the compound and identified the number of components in the mixture based off the differences in polarity. A thin plate was used in TLC that was backed with plastic. Since a “normal phase” chromatography was performed, the plastic was coated with a polar material mixture: silica. Silica was the “stationary phase”. The silica was polar due to the many hydroxyl groups it contained that were readily available for hydrogen bonding. Therefore, the more polar

components of the mixture interacted well with the silica, and the least polar components did not interact as well with the silica. A solvent (7:3 hexanes: acetone) was then introduced to the thin plate. The solvent was known as the “mobile phase,” and was less polar than the stationary phase. Each component interacted differently with the mobile phase and stationary phase. Less polar components interacted better with the mobile phase, while more polar components interacted better with the stationary phase. The capillary actions of the plate allowed the solvent to travel up the plate. As it encountered the mixture, the different components travelled at different rates. The solvent carried the less polar components in the compound. These components began to move up the column reacting more with the solvent that the silica. The more polar components interacted with the silica and thus the solvent had a hard time disrupting the interactions. As a result, the more polar components stayed put or moved very little up the column. The components in the middle interacted with both the silica and the solvent, so they moved up the column but at a slower rate. It was concluded that Xanthophyll, Chlorophyll A & B, Pheophytin B, and Carotene were the components that were in the extract. They contained Rf values of 0.09, 0.20, 0.31, 0.32, 0.41, 0.49, and 0.86. The wet column chromatography was used to isolate and separate the components of the mixture. This technique utilized the same principles as TLC, but instead the mixture was placed on top of the column. The silica gel slurry acted as the stationary phase and the two solvents acted as the nonpolar mobile phase. As the mixture was added to the column, the least polar components travelled the fastest due to the interactions with the mobile phase. Thus, they eluted from the column first. The more polar components interacted with the silica, and as a result, the solvents had a hard time disrupting the interactions. Thus, the more polar components took a longer time to travel down the column and were the last to be eluted. TLC was performed on each fraction to determine the purity and identity of the compounds. Each fraction suggested that Carotene, Pheophytin B, Chlorophyll A & B, and Xanthophyll were present.

Post-Lab Questions: 1. Diethyl ether is the best option. Isopropanol and acetone will dissolve the KCl and will interact with the H2SO4 and create unwanted products. 2. The density of each component determines which is the upper and lower layer. Since the density of the organic layer is less than the aqueous layer, the organic layer rises to the top. 3. Add water! If the water dissolves that indicates the aqueous layer. If it passes through that indicates the organic layer. 4. In the 3:1 ratio, the dots are all at the top indicating that it is too polar. In the 1:4 solution, all the dots are at the bottom indicating that the solution is not polar enough. The 4:5 ratio would be ideal and should be tried next. 5. The addition of more ethyl acetate increased the polarity. He should have added more pentane, which was nonpolar, and then gradually add the ethyl acetate.

6. answered on paper 7. The partition coefficient is the ratio of solubilities of the two solvents. Math shown on paper. 8. This can be possible if both components have the same polarity. Or the other dot cannot be seen under the naked eye and requires a UV light. Adding a more polar solvent may help to separate the two components or utilizing a UV light to see if the other spot is hidden....