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Lee 1 Sarah Lee (UIN: 670332154) CHEM 233 - McQuade 24 June 2014 Lab 2: Chromatographic Methods Separation of Dyes and Spinach Pigments I. Methods and Background The objective of the lab was to determine the separation of dyes and spinach pigments through two different chromatographic methods. Chromatography is the process of separating a chemical mixture into two immiscible phases: a mobile phase and a stationary phase (Gilbert 175). This allows for the extraction of a pure substance. In this lab, both column chromatography and thin layer chromatography were explored. The purpose was to ultimately figure out the relationship between solvent polarity and eluting power. The first method that was performed in the lab was column chromatography, which was used for the separation of two dyes. The goal for using column chromatography was to determine which solvent caused maximum separation of the dye mixture. The second method that was performed was thin layer chromatography (TLC), which was used to separate pigments in spinach leaves. The goal for using TLC was to determine which ratio of acetone and hexane would create the mixture that separated the spinach pigments the best. Column chromatography is a type of solid-liquid absorption chromatography, which means that there is a solid stationary phase and liquid mobile phase (Gilbert 184). To conduct column chromatography, a solid adsorbent is first packed into a column. Then, the mixture to be separated is added to the column followed by an eluting solvent (eluent) (Gilbert 185). When the added solvent/eluent runs through the column, a part of the mixture is bound to the stationary phase while the other is bound to the mobile phase, causing a separation in the mixture. The separation causes the part bound to the mobile phase to elute down the column. The time it takes for the components of the mixture to go through the mobile phase is called the retention time. Retention time is explained by polarity. A highly polar compound would be more bound to a polar stationary phase while a highly nonpolar compound would elute down the column in the mobile phase. In order to move a highly polar compound through the stationary phase, a highly polar solvent would be required. Therefore, when a polar solvent is added to the mixture, a less polar compound would travel down the column quicker. A challenge in column chromatography is that if the solvent is too polar for the adsorbent and the mixture that needs to be separated, the mixture could elute too quickly and the separation would be difficult to measure. In this lab, the adsorbent used was silica gel, but other compounds such as alumina would be appropriate choices as well. The elutropic series of eluting power and polarity of solvents are shown below:

Lee 2 This order represents how polar the solvents are as well as how well they can elute the mixture. As polarity of the solvent increases, so does the eluting power of the solvent. Therefore, as the solvent increases in polarity, it will elute the compound faster (Gilbert 179). This is important because is demonstrates how polarity is involved in column chromatography through the concept of “polar dissolving polar.” It is because of this concept that the mixture is able to separate during the mobile phase. In this lab, the mixture contains two dyes: methylene blue and methyl orange (see Figure 0.0 below). Column chromatography was used in order to separate the mixture in order to see the methyl orange separately from the methylene blue. Silica gel was used as the adsorbent, and a variety of solvents were used, including hexane, 2-propanol, acetone, ethyl acetate, and dichloromethane. These solvents all have varying dielectric constants and thus differing levels of polarity.

Figure 0.0: Structures of Methylene Blue and Methyl Orange The second part of the lab involved TLC. TLC is also a solid-liquid adsorption type of chromatography (Gilbert 176). Like column chromatography, TLC involves a mobile phase and a stationary phase. The stationary phase involves a solid while the mobile phase involves a liquid (Gilbert 172). The stationary phase is called a TLC plate, and it has two notable features: the sample spot and the solvent front. The sample spot is where a small amount of the sample in question is placed, while the solvent front is where the solvent stopped on the TLC plate. The TLC plate is placed in a closed jar called a developing chamber that has a small amount of solvent in it (Gilbert 173). The solvent ascends in the TLC plate and components of the sample are carried up the plate. The polarity of the solvent helps elute the individual components of the sample and separate them (Gilbert 177). As a result, the components are separated and resemble little dots on the TLC plate. In this lab, TLC was used in order to separate several different pigments within a sample of spinach leaf. Like in the column chromatography portion of the lab, the adsorbent material in this lab was silica gel on the TLC plate. However, the solvent used was a combination of hexanes and acetone. The ratio mixtures of acetones and hexanes included the following: 0:100 (no acetone, all hexanes), 20:80 (20% acetone, 80%

Lee 3 hexanes), 40:60 (40% acetone, 60% hexanes), 60:40 (60% acetone, 40% hexanes), 80:20 (80% acetone, 20% hexanes), and 100:0 (all acetone, no hexanes). These varying ratios of both a polar and non-polar solvents were used to find the best ratio for pigment separation on the TLC plate. The pigments to be determined by TLC were chlorophyll a and b (see Figure 0.1), pheophytin a and potentially b, xanthophylls, and carotenes. On the TLC place, chlorophyll a is dark green, chlorophyll b is light green, pheophytins are gray, and xanthophylls/carotenes are yellow to orange.

Figure 0.1: Structures of chlorophyll a and b Also similar to column chromatography is that the results of TLC are also highly dependent on the polarity of the mobile phase. This is due to the fact that each pigment has a different polar affinity to the TLC plate, and so a lower polarity is indicative of lower adsorption. Furthermore, TLC also follows the elutropic series for solvents, and as a result a polar solvent has high eluting power. When using this solvent, less adsorbed pigments on the TLC plate are moved up a higher distance with the solvent. This distance traveled by the pigment is measured using a value called a pigment retention factor (Rf). This value is the distance traveled by the pigment divided by the distance traveled by the solvent, and it is ultimately a measure between the polarity of the solvent and the pigment. Therefore, as a solvent’s eluting power increases, so would the Rf of a pigment. Thus, marks on the TLC plate reflect the retention time and retention factor and emphasize the importance of polarity. A challenge to this experiment is that a very high eluting power would cause all of the pigments to travel to the solvent front and not separate based on polarity. In order to obtain the most visible spectrum of pigments it was found that a 40:60 mixture of acetones and hexanes provided the greatest result. II. Experimental Procedure This lab consists of two parts: column chromatography and thin layer chromatography. The procedure for the column chromatography is as follows:

Lee 4 Column Chromatography: Five pipettes columns were obtained and a small amount of cotton was added to the bottom of each pipette to prevent any spillage of the adsorbent material out of the column. Each pipette was placed in a test tube rack. Next, the pipette was filled to about the two-thirds (2/3) with adsorbent silica gel material. Following that, about two to four drops of the methyl orange/methylene blue mixture was added to each of the five pipettes. Next, a small amount of sand was placed in each pipette. The purpose of the sand is to prevent any disturbance of the stationary phase when the solvent is added. Each pipette was then eluted with one of the following solvents: 2-propanol (ε = 20.2), ethyl acetone (ε = 6.1), acetone (ε = 20.1), dicholoromethane (ε = 9.1), and hexanes (ε = 2.0). (See Figure 0.2 for sample filled pipette.) Afterwards, each column was observed for the amount of separation of the mixture and the effects of the eluting solvent.

Figure 0.2: Setup for Column Chromatography TLC The second experiment conducted in this lab was thin layer chromatography. To begin, three spinach leaves were acquired and ground into a homogenous liquid-like substance with a mortar and pestle. Then, 10 mL of dichloromethane was added to the spinach and mixed very gently. A vacuum filtration system was then setup, which required a Buchner funnel, pressure tubing, a filter adapter, and an Erlenmeyer filter flask. To set this up, the tubing was connected to the vacuum pump on the faucet and was also connected to the tube of the Erlenmeyer filter flask. A filter with filter paper was placed on the opening of the flask. Finally, the Buchner funnel was placed on the top of the setup. The contents of the spinach mixture were poured in the filter and the vacuum sucked the liquid into the flask. This liquid was collected and placed into a separatory funnel, which was sealed with a penny stopper. In this funnel, about 5 mL of H2O was added to the solution. The funnel was swirled gently to separate the aqueous layer from the organic layer. Pressure was released by removing the penny stopper on the funnel. Next, the organic layer was separated from the aqueous layer by emptying it in a flask. The aqueous layer was disposed of while the organic layer was kept. These last few steps were repeated two more times by adding more water, swirling gently, releasing pressure, and separating the organic layer from the aqueous layer. This was done until there was very little aqueous

Lee 5 layer. In the small flask that held the remaining organic layer, small amounts of Na2SO4 was added and mixed well until the solid stopped clumping. This allowed the water to dry out in the sample. The liquid was then decanted into a test tube. After obtaining the sample liquid from spinach leaves, the TLC plates were prepared. Six TLC plates were marked approximately 1 cm from the bottom wit ha base line. A dot was drawn on the base line to represent where the sample would start. A solvent front was also drawn. In addition, six beakers were filled with the acetone/hexane solvent. Each beaker was filled with a different ratio of acetone and hexane. The ratios consisted of 0:100 (no acetone, all hexanes), 20:80 (20% acetone, 80% hexanes), 40:60 (40% acetone, 60% hexanes), 60:40 (60% acetone, 40% hexanes), 80:20 (80% acetone, 20% hexanes), and 100:0 (all acetone, no hexanes). 10 mL of the solvent mixture were poured into each beaker. Using a capillary tube, the sample of spinach mixture was carefully extracted and a few drops were applied to the TLC plates. The TLC plates were then placed in the beakers and then covered with a watch glass in order to create a developing chamber for the TLC (see Figure 0.3). After the solvent reached the solvent front, the TLC plates were taken out and observed.

Figure 0.3: TLC Chamber setup

III. Data Acquisition The data collected included the observations of the column chromatography and TLC. Fr column chromatography, it was observed whether or not the mixture was separated. The solvents used were also observed to determine which solvent separated the mixture the best. The data for TLC was observed to see which acetone/hexane ratio best separated the pigments. The separation data was also collected to figure out which pigments showed up on the TLC plates. The retention factor was then calculated for each TLC plate.

Lee 6 Column Chromatography Data The data obtained for the column chromatography included several observations. These observations were based on the type of solvent/eluent added as well as the degree of separation for the different compounds. (Note: ε = Dielectric constant) Table 1: Observation of Methylene Blue-Methyl Orange Column Chromatography

Trial 1

Solvent System 2-propanol (CH3)2CHOH (*ε = 20.2)

2

Ethyl Acetate CH3-COO-CH2-CH3 (ε = 6.1)

3

Acetone (CH3)2CO (ε = 20.1)

4

Dichloromethane CH2Cl2 (ε = 9.1)

5

Hexanes C6H14 (ε = 2.0)

Observations As soon as the solvent was placed in the pipette column, the separation between methyl orange and methylene blue occurred very quickly. The methyl orange was easily eluted and identified as a light orange color. Methylene blue remained in the stationary phase and was identified by dark blue coloring. There was a poor level of separation when the eluent was added. The methyl orange did separate but only went partially down the column and was stuck in the silica gel. However, more separation was seen here than when the solvent was hexanes. The methyl orange was a light orange color while methylene blue was a dark blue color. Methylene blue stayed in stationary phase. When the solvent was added, the methyl orange and methylene blue immediately separated. The methyl orange was eluted from the system, while the methylene blue stayed in the stationary phase. Maximum separation took place with acetone. Very little separation of methylene blue and methyl orange occurred when the solvent was added. But the methyl orange only eluted about halfway down the way down the column. Methylene blue was in the stationary phase. Very, very low level of separation. Once solvent was put in, as it eluted, the methyl orange started to separate but then almost immediately stopped. The methyl orange and methylene blue stayed at the top of the stationary phase. Methyl orange was light orange and methylene blue was dark blue color. Methylene blue was bound to stationary phase throughout the entire elution.

Lee 7 Figure 0.4: Column Chromatography for the Separation of Methyl Orange and Methylene Blue

Trial 1

Trial 2

Trial 3

Trial 4

Trial 5

Key: Dark blue = Methylene Blue Orange = Methyl Orange White = Empty space or silica gel Table 1 lists all the observation from the addition of each differing solvent. This data can be used to qualify which solvent has the highest eluting power and which separates the compounds the best. The column chromatography was run for approximately 20 minutes.

Lee 8 Thin Layer Chromatography Data The TLC data consists of measurements of mobile phase compositions versus the retention factors of the six pigments. Actual measurements from the TLC plate were used to calculate the retention factors. Each system had approximately ten minutes to run to completion Table 2: Mobile Phase Composition Effect on Pigment Retention Factor (Rf) Pigment Retention Factors (Rf) Mobile Phase Composition (Acetone/Hexanes) 0:100

Chlorophyll a 0.476

Chlorophyll b -

Pheophytin a -

Pheophytin b -

20:80 40:60

0.041 0.347

0.327

0.451 -

60:40

0.898

-

80:20 100:0

0.773 0.674

-

Xanthophylls Carotenes -

-

-

-

0.098 -

0.888 -

-

0.878

0.908 -

-

-

Calculation of Pigment Retention Factor (Rf) for Spinach pigments 𝑅! =

[𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑒𝑑 𝑏𝑦 𝑝𝑖𝑔𝑚𝑒𝑛𝑡 (𝑐𝑚)] [𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑓𝑟𝑜𝑚 𝑡ℎ𝑒 𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒 𝑡𝑜 𝑠𝑜𝑙𝑣𝑒𝑛𝑡 (𝑐𝑚)]

-

-

Lee 9 0:100 Distance from baseline = 4.2 cm !.! !" = 0.476 cm Chlorophyll a: 𝑅! = !.! !"

20:80 Distance from baseline = 5.1 cm !.! !" Chlorophyll a: R ! = = 0.176 cm !.! !" !.! !"

Pheophytin a: R ! = !.! !" = 0.451 cm Carotenes*: R ! =

!.! !" !.! !"

= 0.098 cm

40:60 Distance from baseline = 4.9 cm !.! !" = 0.347 cm Chlorophyll a: 𝑅! = !.! !"

Chlorophyll b*: 𝑅! =

!.! !" !.! !"

= 0.327 cm

60:40 Distance from baseline: 4.9 cm !.! !" = 0.898 cm Chlorophyll a: 𝑅! = !.! !"

Carotenes: 𝑅! =

!.!" !" !.! !"

Xanthophylls: 𝑅! = Pheophytin a: R ! =

=0.908 cm

!.! !" !.! !"

!.!" !" !.! !"

= 0.878 cm = 0.888 cm

80:20 Distance from baseline: 4.4 cm !.! !" = 0.773 cm Chlorophyll a*: 𝑅! = !.! !"

100:0 Distance from baseline: 4.3 cm !.! !" = 0.674 cm Chlorophyll a*: 𝑅! = !.! !"

*note: observed colored dot was very light at splotchy; difficult to determine precise color For observations of the TLC plates, the colors that were seen indicated the following: • Green = chlorophyll a • Light green = chlorophyll b • Yellow = carotenes (above chlorophylls) and xanthophylls (below chlorophylls) • Gray = pheophytin a

Lee 10 IV. Conclusion The goals of the lab were to separate two dyes in a mixture and several pigments in a spinach pigments through two chromatographic methods. The chromatographic methods that were utilized were column chromatography and thin layer chromatography (TLC). Additionally, for both parts of the experiment, it was also determined which solvent created the best separation. Column chromatography was used in order to separate the dyes methyl orange and methylene blue from one another. Ultimately, it was determined that a solvent with a higher dielectric constant caused the quickest elution of the methyl orange from the mixture. This supports the idea that a more polar solvent causes faster separation of the dyes (as a higher dielectric constant indicates a higher polarity). Thus, the solvents with a higher dielectric constant had higher luting power, which made the retention time for separation shorter. This caused the separation of methyl orange methylene blue was determined more quickly. The data according to Table 1 and Figure 0.4 supports these conclusions and are presented as follows. In this lab, 2-propanol, acetone, dichloromethane, ethyl acetate, and hexanes were used as the solvents. Among the solvents used, 2-propanol had the lowest retention time. This is because 2-proponal had a high dielectric constant of 20.2 and quickly eluted methyl orange from the mixture. Additionally, acetone showed the same response because it also has a high dielectric constant (20.1)which allowed the separation of the dyes to happen very quickly. The other solvents—which included dichloromethane (dielectric constant of 9.1), ethyl acetate (dielectric constant of 6.1), and hexanes (dielectric constant of 2.0)—caused little to no separation at all because these solvents were not polar. Dichloromethane eluted the methyl orange slightly, but the dye did not go far down the column. Ethyl acetate also separated the mixture poorly. Finally, hexanes had the highest retention time because it had the lowest dielectric constant; as a result, almost no separation between the dyes occurred at all. Thus, ultimately it was determined that polar solvents such as 2-proponal and acetone were much better for separating the two dyes than nonpolar solvents like hexane. Furthermore, our results show that methylene blue is more polar than methyl orange. This is can be seen from the observation that methyl orange readily elutes down the column with an increasingly polar solvent, as it is weakly adsorbed into the silica gel. On the other hand, because methylene blue is more polar, it was bound to the silica gel because it is also polar. Thus, methyl orange could be eluted faster since it was not as polar and would not be bound to the stationary phase. In the end, it was determined that 2-propanol was the best polar solvent with the highest eluting power and separated the methylene blue and methyl orange the best. In the second part of the lab, thin layer chromatography was used in order to separate the pigments in a spinach leaf. The best ratio for pigment separation was found to be 60% acetone and 40% hexanes. This is due to the fact that a highly polar solvent (above 60% 100% acetone) would cause rapid elu...