Extraction of Spinach and TLC Lab PDF

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Experiment 5 Extraction of Spinach Juice, TLC, and Column Chromatography Lab Report: Written by: Manu Nair 343-75 Lab Dates: Online

Objective: This experiment employed the new techniques of microscale extraction, thin layer chromatography, the gravity column, and analyzing eluted fractions of thin layer chromatography to determine presence of products. The various techniques were employed to determine the photosynthetic pigments within common spinach. New methods were learned, and old methods were reinforced. The objective of this lab was to use thin layer chromatography (TLC) based on differences in polarity and column chromatography to separate compounds. This lab will help gain an understanding of the benefits and uses of both techniques of chromatography.

Procedure 1. 3 mL of spinach juice was measured in a graduated cylinder and added to the screwed-cap test tube. 2. 6 mL of pentane was measured in a graduated cylinder and added to the same test tube. 3. The test tube was capped and shaken vigorously for one minute. 4. The sample tube was taken to a centrifuge and placed inside. Another test tube, filled with water, was placed directly across from the sample in the centrifuge so that load management was equal. 5. After running the sample through the centrifuge, the top layer was separated into a 50 mL beaker using a Pasteur pipet and microscale extraction (Fig 1) . 6. The pentane left in the beaker was evaporated by placing the beaker on a hot plate at 95 °C for a few minutes. 7. Two drops of pentane were added to the beaker so that the TLC reading would be more accurate. 8. 5 mL of the TLC-developing solvent (7:3 hexane acetone) was added in the developing chamber. 9. A stopper was placed on the chamber so that solvent vapors could saturate the chamber. The depth of the solvent was noted and made sure to be less than 0.5 cm. It was allowed to sit for 5 min. 10. The TLC plate was prepared. A pencil mark was drawn 1 cm from the bottom of the plate on the rough silica side. This was made to be the designated start line. The finish line was drawn 5 cm away from the start

Observations The spinach juice was a dark green liquid and was quite opaque. After pentane was added and the tube was shaken, the solution looked less dark and more clear-green.

The sample had two clear separate layers after coming out of the centrifuge. One was dark green and one was light green.

After the pentane boiled off, the solution turned from a lighter green to a darker green. This is most likely due to the pigments left in the beaker.

The fumes from the solvent were evident as it was transferred. The chamber became saturated with the vapor.

The silica side was rough and pencil marks were made to designate where the start and finish lines were.

line. A tick mark was drawn in the center of the start line. 11. Using a micro capillary tube, the spinach extract was loaded onto the TLC plate. The sample was placed on the tick mark on the TLC plate. Sample was continuously added to the spot, while maintaining the same size of the spot. The spot was left to dry. 12. Forceps were used to place the TLC plate into the developing chamber. The chamber was capped immediately so that vapors did not escape (Fig 2). Once the solvent reached the finish line, forceps were used to remove the plate from the chamber and the plate was allowed to dry. 13. Spots were visualized using a UV lamp and all spots were recorded. Using a pencil, an outline of each spot on the plate was drawn.

Spot became very concentrated due to repetitive addition of spinach sample.

The solvent started migrating quickly up the TLC plate.

New Techniques:

Predictions: Polar Pigments in Spinach Extract: Chlorophyll a, Chlorophyll b, Xanthophyll Nonpolar Pigments in Spinach Extract: Beta-carotene

Results: Spot

Color Yellow Green Blue Gray

Distance Traveled 0.9 cm 1.5 cm 1.8 cm 2.3 cm

Rf Value .18 .3 .36 .46

1 2 3 4 5

Orange

4.3 cm

.86

Pigment Identity Xanthophyll (polar) Chlorophyll b (polar) Chlorophyll a (polar) Pheophytin (medium polarity) Beta-carotene (nonpolar) Results Discussion 1:

Results from other lab data: Spot #

Color

Rf value

1st spot 2nd spot 3rd spot

Yellow Green Yellow

0.0847 0.1525 0.8729

Possible identity of compounds Xanthophylls Chlorophyll B Carotenes

Retention Factor is defined as the distance the spot travels divided by the distance the solvent travels. The distance between the start and finish line was 5 cm. The first spot traveled .9 cm, had a yellow color and an Rf value of .18. This is a polar Rf value and the pigment was determined to be xanthophyll. The second spot traveled 1.5 cm, had a green color and an Rf value of .3. This is a polar Rf value and the pigment was determined to be chlorophyll b. The third spot traveled 1.8 cm, had a yellow color and an Rf value of .36. This is a polar Rf value and the pigment was determined to be chlorophyll a. The third spot traveled 2.3 cm, had a gray color and an Rf value of .46. This is an Rf value of medium polarity and the pigment was determined to be pheophytin. The fifth spot traveled 4.3 cm, had an orange color and an Rf value of .86. This is a nonpolar Rf value and the pigment was determined to be Betacarotene.

Results Discussion 2: The first yellow spot had an Rf value of .0847. This is a polar Rf value and the pigment was determined to be xanthophyll. The second green spot had an Rf value of .1525, another polar Rf value. The spot was determined to be chlorophyll b. The final yellow spot had an Rf value of .8729, which is a nonpolar Rf value. The spot was determined to be a combination of carotene pigments.

Discussion of Experiment and Results: Microscale extraction was used in this experiment in order to separate the photosynthetic pigments of spinach. These pigments had a wide range of polarity and were separated using relative Rf values. The order in decreasing Rf was shown as carotenes, pheophytin A, pheophytin B, chlorophyll A, chlorophyll B, and xanthophylls. Rf value is calculated by dividing the distance traveled by the spot (pigment) by the distance traveled by the solvent (5 cm in the experiment). Spots or pigments with lower Rf values are more polar than ones with higher Rf values. This is because, at the molecular level, the polar pigments are more attracted to the silica TLC plate than the nonpolar solvent and the intermolecular attractions between two polar compounds are stronger than a polar and a nonpolar substance. This means the greater the polarity of a pigment, the less distance it will travel due to the polar interactions between its polar functional groups and the TLC plate. This is depicted in the image below:

The solution of spinach pigments was created by grinding spinach with acetone and hexane. The hexane was used to create a solubility environment different from that of water. This helped separate the pigments from any unwanted substances in the experiment. Acetone was used to convert the pigments into the aqueous form due to it being a strong solvent. Thin Layer Chromatography has both a polar stationary phase and a mobile phase with varying polarity, usually a nonpolar substance. The polar stationary phase formed dipole-dipole interactions with the more polar pigments while the moving mobile phase was relatively nonpolar and was able to move the nonpolar pigments along with it. The most nonpolar pigments moved the furthest or fastest as these pigments lacked any polar interactions, such as dipole-dipole interactions, to help it remain attracted to the silica TLC plate. The silica layer was the stationary phase for this chromatography experiment. The plate also provided a medium for capillary action to allow the nonpolar mobile phase to rise up the TLC plate, due to its absorbance. The mobile phase in this experiment was the developing solvent, 7:3 Hexane-Acetone. This was a nonpolar solvent and allowed the chromatography experiment to provide accurate results by bringing the nonpolar pigments along with it, while leaving behind the polar pigments. These results were shown by the different final positions of the spots.

Part 2: Procedure Observations 1. The column chromatography apparatus was set up (Fig 3). The stopcocks were checked to ensure they were shut tight. 2. A cotton plug was placed into the Dr. Davison uses a small amount of cotton; column using a copper wire. A layer of not enough to block the flow and not too little sand was added above the cotton plug to accelerate the flow. (approximately 1 cm). 3. 25 mL of petroleum ether was added to the column. Then, 5 g of fine alumina was added to the petroleum ether. The column was packed by repetitively adding tape to the sides of the column. Fine alumina is a very fine grayish-white 4. Approximately 1 cm of sand was powder added to the top of the fine alumina. Then, the column was drained until the liquid level dropped to the top part of the sand. Drip rate was too slow indicating too much 5. Approximately 0.100 g of Fluorene, cotton may have been used. The compound Fluorenone was placed in a test tube. also did not dissolve immediately. It dissolved 0.5 mL of petroleum ether was added after adding the dichloromethane drop-byto this test tube with the Fluorene, drop. Fluorenone. Dichloromethane was added drop-by-drop in order to dissolve the compound. 6. The sample in the test tube was poured into the top of the column after the petroleum ether drained to the A solid residue is present level of the sand. A watch glass was used to monitor whether Fluorene was leaving the column. 7. Very little Fluorene emerged from the column, so the tip of the stopcock was Very little Fluorene came out of the column washed off to clean all the Fluorene off the tip of it. 8. A chem wipe was also used to clean off the tip so that no Fluorene is contaminating the newly created sample. 9. 10 mL of dichloromethane was added The material moved down the column much quicker. into the column. This helped move material further down the column. Then, a waste flask was used to collect

the liquid. This was done until the second ring moved further down the column. The flask was switched out once the ring dropped down to the cotton plug level. 10. The flask containing petroleum ether was gradually poured onto a watch glass, underneath the fume hood. This process was repeated with methylene chloride and the Fluorene on top of a clean watch glass. Finally, the solid compounds that accumulated on the watch glass were scraped off and weighed.

The solution was yellow and was dripping very slowly. The flasks were switched as soon as the yellow solution touched the cotton swab.

The ether evaporated very quickly

New Techniques:

Results: The video did not provide numerical results. Dr. Davison completed the experiment successfully and was able to separate the two compounds of Fluorene and Fluorenone. This means he was able to take the masses and obtain a percent yield.

Discussion of Results and Experiment: In the first part of this week’s lab, we used Thin Layer Chromatography to separate pigments in spinach based on their polarity. Chromatography, however, is a term used to describe any technique where the components of a mixture are separated by movement through a medium. The rates and final positions of these movements help determine what the identity of the compound is. In Column Chromatography, the components of the mixture are placed down a column with a stationary phase and separated through movement using a mobile phase. In this experiment, the goal was to separate a mixture of Fluorene and Fluorenone, based on their polarity. The compound mixture was passed through an absorbent stationary phase. In this lab, fine alumina was used to fill this role. Fine alumina is polar and acts a good stationary phase by forming dipole-dipole interactions with polar substances and not attracting nonpolar substances. This, like TLC, forces the nonpolar compounds farther away and keeps the polar compounds relatively closer. The difference is that, in column chromatography, the analytes (Fluorene and Fluorenone) flow down a column instead of up a TLC plate. In this lab, before passing the analytes through the column, they had to be dissolved in the mobile phase, or eluent. The eluent used in this lab was petroleum ether. A few drops of dichloromethane had to be added so that the compound would dissolve in the mobile phase. The basics of Column Chromatography involve using gravity and polar interactions to separate the analytes involved. The nonpolar eluent, petroleum ether, forms nonpolar interaction with the more nonpolar compound and brings it down the chromatography column. While this occurs, the polar compound forms stronger polar interactions (dipole-dipole) at the molecular level with the polar stationary phase, fine alumina. This causes the separation of the two compounds and the nonpolar compound is eluted first out of the column. In the video, it was determined that Fluorene was the more nonpolar compound due to it passing much quicker through the column than Fluorenone. This was also reinforced by the residue that was left on the watch glass. Fluorenone was the more polar compound and therefore passed much slower through the column. This required the addition of dichloromethane, a polar compound, to increase the flow rate of Fluorenone. After the rest of the Fluorenone passed through the column, the two compounds were successfully separated. The main difference between column chromatography and TLC is that column chromatography is more useful in separating two compounds in a mixture, while TLC is more useful for identifying compounds based on their Rf values. The two techniques have many applications and it is important to identify your objective in an experiment before choosing which technique to employ.

Post Lab Questions: 1. The best solvent to use would be diethyl ether since it is nonpolar and would not form interactions between the ions of KCl. Diethyl ether is a good choice because it would not react with the sulfuric acid. Isopropanol is not a good choice as well as it would react and become deprotonated by the sulfuric acid. The polarity of acetone would make it a less likely choice as it would interact with the KCl ions. 2.

a. The organic layer is the darker top layer, while the aqueous layer is the bottom lighter layer. The main physical factor that determines where each solution goes is density. Organic solutions with lower density than aqueous layers would sit on top of them. b. The bottom layer would be byproducts that dissolved into the water which are now waste. The top would be the main product dissolved in diethyl ether. Finally, the air filling the space would most likely be carbon dioxide byproducts. 3. Water can be added to the separatory funnel to test which layer is aqueous. The water would sink into the bottom layer if the top were not aqueous. The water would dissolve and not sink to the bottom if the top layer was aqueous. 4. The 1:4 ratio of ethyl acetate to hexane was too nonpolar as the spots were localized at the bottom of the TLC plate. The 3:1 ratio of ethyl acetate to hexane was too polar in this situation as the spots were localized near the top of the plate. A 2:3 ration of ethyl acetate to hexane would be best to ensure that the spots localize near the center. 5. Darcy made the mistake of using polar ethyl acetate to create his silica gel slurry. This created a stationary phase that was too polar. The solution to this mistake would be to use pentane to create the slurry instead. 6. From top to bottom: A, D, C, B. 7. Benzoic acid solubility: 10g/100mL diethyl ether and 1g/100mL water. V=300mL, m=1.8g benzoic acid, molar mass=122g/mol Kp=? Ether: 10 g/(122 g/ mol) x 1000 = .82 m Water: 1g/(122 g/ mol) x (1000/100 mL) = 0.082 m Log(Kp) = 0.82/0.082 = Kp = 10 q = (300/(300+(10x40))³= 0.08 8. The spots appeared to be one as they contained similar polarities. This can be resolved by

creating eluent solutions of varying polarity to discover the correct ratio that would allow for separation. A more nonpolar solution would be used at first and then adjusted to allow one of the fractions to flow faster than the other....