Lab Report 2 - Lecture notes 2.22 PDF

Preview

Summary

Lecture reading from TA...

Description

Lab 2: Chromatographic Methods: Separation of Dyes and Spinach Pigments by Column and TLC. Zeba N. Siddiqui (Partner: Keti Berberi) September 19, 2014

Methods and Background Two experiments were performed to test the separation of pure compounds from a mixture, which is known as chromatography. Column Chromatography is one of the experiments and its goal is to create an effective column chromatography setup and procedure that will separate methyl orange from methylene blue. Variables of the procedure includes the amount of silica used, the construction of the model, and the composition of the mobile phase. Thin-Layer Chromatography (TLC) is the second experiment. Its primary goal is to develop mobile phase composition. This will separate five pigments: chlorophyll a, chlorophyll b, pheophytin a, carotenes, and xanthophylls. Pigments of spinach leaves will be used with the TLC plate and an understanding of polarity, retention time, and retention factor is necessary to complete the analysis of the results. Separation of the compounds is due to the affinity to polarity of the solvent to the substance. A stationary phase is a solid, or liquid, that does move through the TLC plate, but allows liquids to flow. Mobile phase, however, is a liquid, or gas, that flows through the stationary phase. Compounds with a higher affinity towards the stationary phase (polar) will interact with the solid, preventing movement. Compounds that lean towards the mobile phase (nonpolar/ less polar) will interact with the liquid, which allows movement to flow through the stationary phase. Column Chromatography is the technique used to purify compounds from mixtures using a pipette column. This method is used for larger sample sizes. Dye mixture is added to a solvent, a separation is visible after an adequate amount of time is given, an example of the setup is shown

in Figure 1. A more weakly absorb compound is eluted faster from the column than a more strongly absorbed compound. TLC is also helps separate mixtures into their individual components. This method is used with smaller sample sizes and uses TLC plates. These plates are thin sheets, one side coated with silica gel and the other side layered with aluminum. Drops of spinach extract are placed on the plates and after, placed into a mixture of mobile phase compositions. The solvent begins to rise up, which shows the separation of the pigments within the extraction. An example of this is depicted in Figure 2. However, the more polar the sample is, the slower it will move up the plate; consequently, the less polar, the faster the compound leaves exits the plate.

Figure 2 TLC

Figure 1 Column Chromatography

Polarities are important in determining how well a pure substance separates from a mixture. Silica gel contains an alcohol (OH) functional group making it a polar molecule. As a result, any polar molecule that comes across the silica gel will attach to it and become immobile. On the

other hand, if a nonpolar solvent is used, then nonpolar molecules will interact with the nonpolar solvent and travel through the stationary phase, the silica gel. The ability of a solvent to move a compound through a stationary phase and not allow it to be absorbed by the solid is defined as a solvent’s eluding power.

Experimental Procedures: There are six steps to follow for experiment one, Column Chromatography. The first is to prepare five pipette columns, supported with five test tubes, for dry packing. Dry packing is done by first stuffing a piece of cotton at the opening of each pipette, using a piece of copper wire to bring it all the way down the length of the pipette. This allows the stationary phase to stay in the column, preventing it from falling out. Once the cotton is in place, a pinch of sand is added above the cotton, then add a good amount of the absorbent, silica gel, in a tightly packed manner. Step two and three can be done in any order. One step is to add another pinch of sand above the absorbent then add two to four drops of the 1:1 mixture of methylene blue-methyl orange; or adding the 1:1 mixture then the pinch sand. Step four is to elute each column with any five solvents of choice, found under the hood. Step five is to collect and observe the colorings of the pipettes. Finally, step six is to repeat until the goal is meet for each solvent and column. Results and interpretations can be found on Table 1: Column Chromatography Data Table of Solvents and Observations. Experiment two, TLC, consists of two procedures, the actual lab (TLC) procedures and the extraction procedures. There are eleven steps for the extraction. The first is to obtain and grind the spinach leaves in a motor. Grinding is complete once the leaves are homogenous. Then add 5mL of methylene chloride to the mixture and mix. After that, a filtration of the mixture is to be

performed using a vacuum filtration. The next steps indicated that the collected liquid, the filtrate, is to be gathered into a separatory funnel. Then add 5mL H2O in the funnel. Next, drain the organic layer from the funnel into a flask, the aqueous solution can be discarded. Pour the organic layer back into the separatory funnel. Add 25-50mL of H2O to wash the organic layer, then repeat the last few steps (adding the 5mL of H2O-draining organic layer). After, place the organic layer into a 25-50mL Erlenmeyer flask. However, if you have too little organic layer, add an additional 5mL of methylene chloride. Then, add small quantities of Na2So4 until there are no longer any clumps. Step ten is to transfer the solution into a clean test tube. Finally, concentrate the solution by evaporating about half of the methylene chloride in the fume hood. Solution goes from light green to a darker green. The TLC procedure consists of eight steps. Step one is to obtain TLC plates and mark them with a pencil a centimeter above the bottom. This will represent where the pigments began their travel up the plate. Step two is to transfer some of the extract to a capillary. Step three is to apply the extract to the plate on top of the spot drawn. The extract should be visibly seen for an accurate application. Next, the plate must be placed into a beaker tall enough to cover the plate completely and enough room to put a watch glass to seal in the plate to prevent evaporation of the extract. Step five is to remove the TLC plate from the chamber when the elution has reached approximately 0.5-1.0cm from the top of the plate. A line needs to be drawn to make the conclusion of the solvent’s travel. Step six is a repeat of step four to complete a table of different mobile phase compositions (Acetone: Hexane- 0:10, 2:8, 4:6, 6:4, 8:2, and 10:0). Step seven is the decision of which two TLC plates provided the best separation of all five pigments (chlorophyll a, chlorophyll b, pheophytin a, carotenes, and xanthophylls), six if pheophytin b can be observed. Step eight is to repeat step seven until complete separation of all five pigments are

observed. Results can be found on Figure 3: Results of TLC Plates and Table 2: Pigment Retention Factors (Rf).

Data Acquisition: Experiment 1: Column Chromatography Trial 1

Solvent System Methanol (Ef of 32.6)

2

Hexane (Ef of 2.0)

3

2-Propanol (Ef of 20.2)

4

Ethyl Acetate (Ef of 6.0)

5

Acetone (Ef of 20.7)

Observations -Very top is a blue/green band. -Below the green is a thick band of yellow that takes up half the pipette. -Below the yellow is a very little amount of orange. -Rest of pipette was untouched by the solvent -Very top is a blue/green band. -Very thin band of yellow below the green. -The rest, from below the yellow to the end of the pipette, is clear, reaching all the way down the pipette. -Very top is a blue/green band. -Thick band of yellow below the green that takes up half the pipette. -Gap between the yellow and the rest of the absorbent, solvent did not reach the rest of the pipette. -Very top is a blue/green band. -Medium band of yellow below the green that takes up a quarter of the pipette. -The rest is a clear color where the solvent reached the end of the pipette without leaving color behind. -Very top is a blue/green band. -The rest of the pipette is completely yellow.

Table 1 Column Chromatography Data Table of Solvents and Observations

In Table 1, the results if the experiment is observed. Each trial represents each pipette column. Each pipette was observed to contain a thin ring of blue/green at the top of the column. This is due to the separation of the 1:1 mixture of methylene blue-methyl orange, methylene blue being more polar. The orange that was found traveling down the column was methyl orange. The first

trial is Pipette 1, containing Methanol, with an Ef of 32.6, as its solvent. It was observed to have a thick layer of yellow and a tiny ring of methyl orange. Separation of the compounds were pure, where at least half of the column revealed color and expressing a mobile phase composition. The affinity of the solvent towards the dye was strong, where the elution was slow to reach the end of the pipette. Trial two represented Pipette 2 which contained Hexane, with an Ef of 2.0, as its solvent. A tiny band of yellow was present. The separation was not pure and remained in the stationary phase, only letting the liquid pass through. Trial three was Pipette 3, containing 2Propanol, with an Ef of 20.2, as its solvent. It was observed to have a thick band of yellow that reached half way down the column. Separation was possible, the solvent was pure, but the affinity was weak. Trial four was Pipette 4, containing Ethyl Acetate, with an Ef of 6.0, as its solvent. This too, like Pipette 2, was observed to be immobile, yet it was less polar than Pipette 2 because the band of yellow was thicker in Pipette 3 than in Pipette 2. The liquid passed through the column, while the solid stayed secure. Finally, trial five was Pipette 5, where Acetate, with an Ef of 20.7, is the solvent. This revealed a complete separation of the pure compound. The yellow band reached down the column entirely. Experiment 2: TLC Retention Factor (Rf) = distance traveled by substance distance traveled by solvent Rf (2:8): = 14cm = 0.50(pheophytin a) 28cm

= 16cm = 0.57(chlorophyll b) 28cm

= 18cm = 0.64(chlorophyll a) 28cm

= 3cm = 0.09(chlorophyll b) 35cm

= 8cm = 0.23(pheophytin a) 35cm

Rf (4:6): = 1cm = 0.03(chlorophyll a) 35cm

Figure 3 Results of TLC Plates

Mobile Phase Composition (Acetone/Hexanes ) 0:10 2:8

Chlorophyll a

Chlorophyll b

Pheophytin a

0 0.5

0 0.57

0 0.64

4:6

0.03

0.09

0.23

Pheophytin b (may not be observed) 0 Not observed Not observed

Xanthophylls

Carotenes

0 Not observed Not observed

0 Not observed Not observed

Table 2 Pigment Retention Factors (Rf)

Table 2 expresses the results of the TLC Plate. It contains the two most separated compositions, which were the 2mL of Acetone mixed with 8mL of Hexanes and 4mL of Acetone mixed with 6mL of Hexanes. The bands closet to the original dot are more polar, which make them attached and attracted to the stationary solid, the silica gel. 0mL of Acetone mixed with 10mL of Hexanes is the stationary phase of all the compositions. The rest are mobile phase compositions with varying distances. Chlorophyll a is the lighter shade of green found on the plate, chlorophyll b is the darker green, normally found above chlorophyll a, pheophytin a is the gray colored band, and carotene is a pale yellow colored band. In the 2:8 mixture, three bands where observed, chlorophyll a, chlorophyll b, and pheophytin a. With a total distance of 28cm, chlorophyll a moved 18cm up the plate from its starting point. The Rf value is 0.5. Chlorophyll b was 16cm away from the starting point. Chlorophyll b was discovered to be more polar than chlorophyll a.

Its Rf value is 0.57. Pheophytin a traveled a total 14cm, being the most polar of all three bands. Its Rf value is 0.64. The 4:6 mixture had the same pigments found in the 2:8 mixture, however, it consists of varying Rf values and the total distance traveled by the solvent, but the order of polarity is the same, pheophytin a being more polar than the chlorophylls.

Conclusion: The purposes behind the chromatographic experiments was to separate and extract pure pigments from mixtures. Thin-Layer and Column Chromatography were used to achieve this goal. Polarity and the affinity the solvents (mobile phase) had towards the silica gel (stationary phase) were very important in the separation processes. Polar solvents had a higher affinity to the silica gel because the silica gel has an alcohol functional group making it polar. With higher polarity, polar molecules attract to polar molecules, it was discovered that the molecule that did not move, the ones that stayed stationary, are polar. The solvents that traveled down the pipette column and the solvents that traveled up the TLC plates were concluded to be nonpolar. Experiment one involved Column Chromatography. It was witnessed that the blue/green dye stayed around at the top of the pipette while the orange dye slowly traveled down, hence, methylene blue is more polar than methyl orange. This is due to the dye pigment’s polarity attraction towards the polar silica gel. The yellow pigments were the solvents color. Those pigments are less polar and will sink. Liquid passed the solid if the solvent is completely nonpolar, as witnessed in Pipette 1 and 2. Experiment two involved Thin-Layer Chromatography. This determined which pigments of the spinach where pure. As the solvents traveled up the plates, the pigments separated and distinction between the pigments where visible. Distances of each pigments travel was calculated, which

allowed determination of polarities. Chlorophylls (green pigments) stayed closest to the bottom, the pheophytin (gray pigment) was around the middle, and xanthophylls/carotenes (yellow pigments) traveled the highest. Based off this observation, it can be concluded that the lower the pigments were, the more polar they are. The opposite is also seen, the higher the pigments traveled up the TLC plates, the more nonpolar they are. In order of decreasing polarity, the pigments are concluded to behave as such: chlorophylls> pheophytin> xanthophyll/carotenes. With the different Acetone/ Hexanes ratios, we were able to view diverse Rf values. The more Acetones there were, the less movement there was, however, the more Hexanes there were, the more the pigments moved away from the starting point. Thus, the smaller the retention factor, the more polar the pigment; the larger the retention factor, the more nonpolar the pigment. Based on the results, it is noted that polarities of certain pure substances can be determined through chromatography and retention factors.

References: Gilbert, J.C., and Martib, S.M., Experimental Organic Chemistry: A Miniscale and Microscale Approach, 4th Edition, Cengage Learning, Boston, MA, 2006. Landrie, C.L., and McQuade, L.E., Organic Chemistry: Lab Manual and Course Materials, 5th Edition, Hayden-McNeil, LLC, Plymouth, MI, 2016....