Lab 2 - Lecture Notes L2 - Chromatography Methods: Separation Of Dyes PDF

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Lab 2: Chromatographic Methods: Separation of Dyes and Spinach Pigments by Column and Thin-Layer Chromatography Milan Patel February 3, 2016 Methods and Background: There were two experiments performed in this lab. The purpose of this lab was to get familiar with techniques such as column chromatography and thin-layer chromatography. In addition, this lab helped to understand the relationship between solvent polarity, given the dielectric constant, and eluting power of each solvent in these techniques. Another purpose of this lab was to identify the effect of functional groups on retention time. This was done by understanding the elutropic series. The column chromatography and thin-layer chromatography, used in this lab helps to observe the effects of polarity of the solvent, eluting power, and adsorbent in the separation of dyes and spinach pigments. In the first experiment, a mixture of two dyes methylene blue and methyl orange were separated using the column chromatography. Whereas, the second experiment was used to separate spinach pigments. Figure 0.1 shows the elutropic series for polar stationary phases. Increasing adsorption on polar stationary phases

RCO2H > ROH > RHN2 > RR’C=O > RCO2R’ > ROR’ > C=C > R-X Figure 0.1: Elutropic series for polar stationary phases. Chromatography is the technique of partitioning solute between two immiscible phases, one stationary and other being a mobile phase. The stationary phase is the fixed solid or liquid phase. In this lab, we used silica gel as a polar stationary phase. The mobile phase is the phase with a liquid or a gas that is passes through the stationary phase. In this first experiment of the lab, we used different solvents in each trials as our mobile phases. Thus, compounds that are more polar will have a greater affinity to polar stationary phases and less polar compounds are less attracted to polar stationary phases. Furthermore, depending upon the solvent, the dyes would travel down the pipette and that’s how the separation would be achieved. In this lab, we were trying to separate methylene blue and methyl orange. Since both dyes are relatively polar, the solvent that would work the best would be the polar solvent as well. However, silica gel, in this particular experiment, has the highest polarity and that is why methylene blue would have higher affinity to the silica gel since methylene blue is more polar than methyl orange. Thus, methyl orange will spend more time in the mobile phase because it is attracted to the mobile solvent which is less polar than silica gel. That is the main reason we saw the separation of the dyes in this first column chromatography experiment. The figure 0.2 shows the two dyes used in this experiment.

Figure 0.2: Methylene Blue (Left) and Methyl Orange (Right). Thin-Layer Chromatography (TLC) is another technique that we used in this lab. This was used in order to separate chlorophyll a and b, xanthophyll, pheophytins a and b and carotenes which are the components of the spinach leaves. In TLC, small plates are used which are consist of a layer of stationary phase bound to the rectangular glass. A small amount of the mixture being separated will be placed on the stationary phase, and the plate is then placed in a chamber containing the eluting solvents. Different solvent compositions will be used to determine which solvent efficiently separates more pigments on a TLC plate. For example, different ratios of Acetone and Hexane will be used to separate the pigments. Acetone is more polar than hexane, thus, the retention factors of the various components in spinach leaves will be based on the affinity of polar components of the mixture to the polar stationary phase. This experiment is slightly different from the column chromatography is that the capillary action moves components upward depending on affinities for the mobile phase. In order to perform the thin-layer chromatography technique we were also required to use vacuum filtration and readymade ThinLayer Chromatography plates. TLC determines the separation of six pigments in spinach leaves. However, none of the TLC plates had more than four pigments distinguished on it which means the data is not significantly right. The most of the pigments observed were chlorophyll a, chlorophyll b, pheophytin a and xanthophyll. One of our plate had four distinct bands there was yellow, green, gray and light gray. Thus, there must be errors occurred while performing the experiment. The possible errors could include dilution of spinach mixture which could lead to the less concentrated pigments. Figure 0.3 has shown the different pigments of the spinach leaves.

Figure 0.3: Six Pigments of Spinach Leaves. Going from left to right: Chlorophyll a and b, Pheophytin a and b, carotene and xanthophyll

Experimental Procedure: This lab consist of two separate experiments. One was Column Chromatography and other being Thin-Layer Chromatography. Thus, first we performed column chromatography and observed the experiments. Secondly, we performed thin-layer chromatography and collected our data with the observations. Column Chromatography: In order to perform this experiment, the five pipettes were prepared, shown in the figure 0.4 with the layer of cotton, sand, silica gel, dyes and sand again. First of all, a ball of cotton was inserted with using the copper wire to push it down the pipette. Then, small quantity of sand was added on the top of the cotton. The columns were filled with ¾ of silica gel in a hood because silica gel is toxic. Once each pipette were filled with silica gel, 4 drops of 1:1 methyl orange and methylene blue were added to the columns. The columns were number for different solvent system in order to keep track of each solvent for each column. For example, number one column was filled with methanol up to the brim twice. Number two column was filled with acetone up to the brim twice. Number three column was filled with 2propanol up to the brim twice. Number four column was filled with ethyl acetate up to the brim twice. Number five Figure 0.4: Column Chromatography column was filled with hexane up to the brim twice. The apparatus. . solvents were collected in test tubes. The observations were recorded that included identification of different colored bands. Thin-Layer Chromatography: Extraction of the pigments from the spinach leaves: Two fresh leaves of spinach were grinded in a mortal with a pestle until they were homogenous. Then, 5 ml of methylene chloride was added to the mixture which was mixed well. Then, the apparatus for vacuum filtration was set up which included air tube connected to Erlenmeyer flask and to the vacuum chamber. Afterwards, a ceramic funnel was prepared with a filter paper and was fixed on the mouth of the Erlenmeyer flask. The vacuum chamber was turned on while the mixture of spinach extraction was added for filtration. The filtrate was transferred into a

separatory funnel. Then 5 mL of H2O was added in the separatory funnel which was then swirled gently until there was a separation of bottom organic layer and aqueous layer. The organic layer was drained into a clean flask and the remaining aqueous layer was discarded. This process was repeated couple extra times. In the end, small quantities of Na2SO4 were added to the solution until no new clumps were being formed. The purpose of adding Na2SO4 was to make the pigment more concentrated and get rid of water as much as possible. Thin-Layer Chromatography Preparation: For this part of the experiment, six TLC plates were numbered and a line was drawn approximately 1 cm from the bottom with a dot on it as a starting point as shown in the figure 0.5. The open end of a capillary tube was immersed into the filtrate which was then applied on to the spot on TLC plate. The filtrate was allowed to evaporate to identify the left spots. This process was done 10-15 times while a green spot was identified easily on TLC plate. Then, six beakers were set up that had different solvents for the experiments. TLC plate one was immersed into 0 ml Acetone and 10 ml Hexane. TLC plate two was immersed into 2 ml Acetone and 8 ml Hexane. TLC plate three was immersed into 4 ml Acetone and 6 ml Hexane. TLC plate four was immersed into 6 ml Acetone and 4 ml Hexane. TLC plate five was immersed into 8 ml Acetone and 2 ml Hexane. TLC plate six was immersed into 10 ml Acetone and 0 ml Hexane. The beakers Figure 0.5: TLC Chamber setup were covered with watch glass to eliminate excess air flow into the system. The TLC plates were taken out from the beakers after the eluent reached the top of the TLC plate and were observed. Then retention factors were calculated by taking the distance the pigment traveled divided by the distance the eluent traveled. Data Acquisition/Presentation: The data collected included the observations of the column chromatography and TLC. For column chromatography, it was observed whether or not the mixture was separated. The solvents used were also observed to see which solvent separated the mixture clearly. Whereas, the data for TLC was observed to see which acetone/hexane ratio best separated the pigments of the spinach leaves. For this experiment, the separation data was also collected to figure out retention factor for the each pigments. -Experiment 1: Column Chromatography 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. Dielectric constants also helped us to figure out the polarity of the each solvent. Data Table 1: Observation of Methyl Orange-Methylene Blue Column Chromatography Trial 1

Solvent System with Dielectric Constants Methanol (32.6)

2

Acetone (20.7)

3

2-Propanol (20.2)

4

Ethyl Acetate (6.1)

5

Hexanes (2.0)

Observations Dyes separated and eluted in a reasonable time. This was very quick, and distinct separation with dark yellow eluent, but blue band stayed on top. Dyes separated but didn’t elute as fast as methanol. The yellow dye traveled down the test tube and tool long time. While the blue band stayed on top, the large yellow portion was seen in the silica gel. Dyes separated better than ethyl acetate. It too little less time compare to ethyl acetate but took longer time compared to Acetone. Dyes separated partially and the eluting time was much longer. Blue color at the top and yellow color was observed throughout the stationary phase. The separation of dyes was not distinct and the eluting time was very high. The eluent in the test tube was colorless suggesting that none of the dyes eluted out from the column.

Data table 1 lists all the observation from the addition of each different solvent. This data can be used to qualify which solvent had the highest eluting power and which separated the compounds the best. Since the silica gel and dyes are both polar, a polar solvent will be very efficient. Methanol has an alcohol group which makes it more polar than the silica gel. Methylene blue is slightly more polar than methyl orange. Due to this difference in polarity, the methylene blue spends more time in the silica gel which is the stationary phase because of the similarity in their polarity and methyl orange travel in the mobile phase along with methanol since they both are slightly less polar than silica gel and methylene blue. Hexanes on the other hand are non-polar, and since “like dissolves like”, the dyes will spend more time in the stationary phase since they are polar and hexane is a non-polar solvent. -Experiment 2: Thin-Layer Chromatography (TLC)

This experimental data consists of measurements of mobile phase compositions versus the retention factors of the six pigments. The retention factor was calculated by using the following equation and data table 2 summaries the different pigments observed in the experiment with their retention factors: Retention Factor (R f )=

Distance Travelled by Substance(dx ) Distance Travelled by Solvent (ds)

The ratios are presented as Acetone:Hexane 0:100

60:40

Chlorophyll b: Rf = 1.1 cm / 4.20 cm = .26

Pheophytin b: Rf = 1.4 cm / 4.6 cm = .30

20:80

Xanthophyll: Rf = 3.5 cm / 4.6 cm = .76

Chlorophyll b: Rf = 1.1 cm / 4.75 cm = .23

Chlorophyll a: Rf = 3.7 cm / 4.6 cm = .80

40:60

Peophytin a: Rf = 3.91 cm / 4.6 cm = .85

Chlorophyll a: Rf = 1.11 cm / 4.49 cm = .25

80:20

Pheophytin a: Rf = 1.51 cm / 4.49 cm = .34

Chlorophyll a: Rf = 5.85 cm / 6.1 cm = .96 100:0 Chlorophyll a: Rf = 5.12 cm / 5.12 cm = 1.0

Data table 2: Mobile Phase Composition Effect on Pigment Retention Factor (Rf) Mobile Phase Composition Acetone/Hexane

Pigment Retention Factors (Rf)

Chlorophyll b .26

Pheophytin a -

Pheophytin b -

Xanthophyll s -

Carotenes

0:100

Chlorophyll a -

20:80

-

.23

-

-

-

-

40:60

.25

-

.34

-

-

-

-

60:40

.80

-

.85

.30

.76

-

80:20

.96

-

-

-

-

-

100:0

.10

-

-

-

-

-

Table of Pigments and their Colors: Pigments Xanthrophyll Chlorophyll a Chlorophyll b Pheophytin a Pheophytin b Carotene

Colors Yellow Green Green Gray May not be visible Light Yellow

Carotenes are non-polar substance so they will not migrate along the plate if they are immersed in a solution of pure acetone, since acetone is polar. In our experiment we were not able to extract carotene due to too much diluted sample.

Conclusion: The main goal of this lab was to understand the techniques of column chromatography and thinlayer chromatography. The experiment of column chromatography helps establish the relationship between the polarity and eluting power. Silica gel was used along with polar and non-polar solvents to separate a 1:1 mixture of methylene blue and methyl orange. Methylene blue is more polar than methyl orange and since silica gel is of highest polarity of all, Methylene blue will have a higher affinity for the stationary phase. Methyl orange is slightly less polar than methylene blue so when using a polar solvent such as methanol it will spend more time in the mobile phase since it will have higher affinity for the polar solvent. Since the dyes are polar, a polar solvent will work best to separate them. Non-polar solvents such as hexanes will not work because “like dissolves like”, and the dyes have higher affinity for the polar substance. Polar solvents such as methanol separated dyes into two different components easily than non-polar solvent such as hexane. In the case where the dyes were separated, the blue color band being the most polar was seen on the top of the stationary phase and yellow eluent was out of the column being the least polar component compared to others. The greater affinity of polar components with the polar stationary phase led blue dye to stay in the stationary phase for the longer period of time. Thus, overall the blue dye had higher retention time and yellow dye with lower retention time. Even though yellow dye, methyl orange, is polar it is less polar than methylene blue in this

experiment. That is the reason we see blue dye to be attracted to most polar silica gel and yellow dye travel down the column along with the polar solvents. Thus, from the experimental result we can see that the methanol works the best due to its higher polarity compared to others. In thin-layer chromatography, the extract from spinach leaves were applied on to TLC plates which were placed into beakers with different ratios of acetone and hexane. In the experiment, the polar stationary phase was the silica gel plate and the mobile phase was the mixture of the two solvents acetone and hexane. However, there was the similar concept used to make conclusions. Acetone is highly polar while hexane is non-polar compounds. Thus, pigments that are polar will be easily seen with the acetone solution and pigments that are non-polar will be seen with hexane. Since carotene is non-polar and chlorophylls, Pheopytins, and xanthophyll are polar the most ideal solvent to be use would be a mixture of acetone and hexane. To determine the difference between chlorophyll a and b since they are both green, one would have to look at the polarity. The one with higher polarity will always be lower since silica is polar and it will have higher affinity for the silica. Chlorophyll b is more polar than chlorophyll a because of the aldehyde group. The best ratio for pigment separation was found to be 60:40 acetone to hexane. This emphasizes that the polar substances can separate the pigments more distinctly than nonpolar substances. It was very hard to observe carotenes in any of the trails in our experiment. The reason behind it could be that our sample was not concentrated enough and we also did not carefully run the experiment. For example, not covering the chamber properly may lead to evaporate the organic compounds and we couldn’t get the significant results. However, from this experiment we learned that that “polar attracts polar” and “non-polar attracts non-polar.” Overall, the goals of this lab were to determine which solvents best separated two dyes in column chromatography as well as six pigments in spinach leaves in thin-layer chromatography. Also, we also learned the importance of polarity in the separation of compounds. Ultimately, it was discovered that eluting power is based on polarity. Thus, a higher eluting power meant a higher polarity. Due to strong polarity, solvents were able to separate compounds well. However, the challenges were faced in thin-layer chromatography. In TLC, we added more of water and methylene chloride in order to collect enough quantity of the sample. But when that sample ran through TLC, the pigments were so light and it was hard to distinguish the pigments. Due to more dilution, we weren’t even able to see some of the pigments on the TLC plate. Another challenge was to make sure that the extract from spinach leaves doesn’t evaporate. Even after being extra careful putting the glass plate on the chamber, some of the solutions had to evaporate due to a small gap between the glass plate and beaker mouth. All in all, after this experiment, we have a better understanding of the relationship between polarity and the elution power. We are now successfully able to separate a mixture of dyes using the column chromatography and be able to separate pigments from spinach leaves using thin-layer chromatography. In addition, we also learned how polarity plays an important role in both of these techniques. References

Gilbert, J.C. and Martin, S.F., Experimental Organic Chemistry, Cengage Learning, New York, 2011, 5th Ed., pp. 179 – 184 and 188 - 192....