Column Chromatography

Column Chromatography Pre-lab Purpose: The purpose of conducting the column chromatography lab is to separate and purify solids and liquids. Introduction: Chromatography is the separation of multiple mixtures into their components as a result of their molecular interactions with a mobile phase and a stationary phase (Keller et al. 66). In this lab the experiment will be focusing on column chromatography which is another method of chromatography. Column chromatography has a stationary phase which is a solid adsorbent, and a mobile phase which is the liquids. The absorbent is placed in a glass column and the liquid phase is added to the top and will then flow down through the column by gravity or an external pressure. The main reason for using column chromatography is used as a purification technique (University of Colorado at Boulder, Column Chromatography). In column chromatography one of the most fundamental concepts that is needed and utilized in this lab for column chromatography is the polarity. The polarity of a chemical can determine the amount of interactions the sample molecules have with the eluent and adsorbent (Column Chromatography). Figure 1. Column Chromatography Setup Figure 2. Part 1: Separation of a Mixture of Ferrocene and Acetylferrocene Figure 3. Part 2: Separation of a Mixture of Nitroanilines Isomers Table of Physical Constants: Name Chemical Formula Formula Weight Dichloromethane CH2Cl2 84.93 Ferrocene C10H10Fe 186.04 Silica SiO2 60.084 Melting Point Boiling Point °-96.7C 39.6°C 172.5°C 249°C 1710°C 2230°C Density Solubility 1.33 g/cm3 Ethyl, acetate, alcohol, hexanes, benzene, diethyl ether 1.11 g/cm3 benzene 2.65 g/cm3 Hot potassium hydroxide and hot sodium hydroxide Hexane C6H12 86.18 Toluene C6H5CH3 92.14 Al2O3 102 Acetone C3H6O 58.08 2-nitroaniline (Ortho-nitroaniline) C6H6N2O2 138.14 4-nitroaniline (Para-nitroaniline) C6H6N2O2 138.12 C12H12FeO 228.07 C5H12O 88.15 Alumina Acetylferrocene t-butyl methyl ether (TBME) -95.35°C 69°C -95°C 110.6°C 2054°C 3000°C -94.9°C 56.1°C 71.5°C 284°C 147°C 332°C 83°C 161°C -109°C 55.1°C 0.661 g/cm3 Ethanol, ethyl ether, and chloroform 0.87 kg/m3 Acetone, alcohol, benzene, chloroform, ether 3.97 g/cm3 N/A 784 kg/m3 Miscible in water, benzene, alcohol, ether, and chloroform 1.442 g/cm3 Ethanol, ether, acetone, hot water 1.44 g/cm3 Methanol, ether, acetone, benzene, chloroform 1.014 g/cm3 Organic solvents 0.740 g/cm3 Water, ethanol, ethyl ether, Procedures: Part 1: Separation of a Mixture of Ferrocene and Acetylferrocene Used hexane to hold the alumina Ferrocene is less polar and Acetylferrocene is more polar 1. Prepare the column as described Acetylferrocene would ravel longer than 2. Use 6in of alumina 3. We're about 0.05 g of Ferrocene since it is more polar ferrocene/acetylferrocene and Dissolve them together in 0.5 mL of CH2Cl2 Open the stopcock to make sure the solvent 4. Run the solvent to just below the top of the sand in the column, and carefully apply the solution ferrocene 5. Drano small amount of solvent to bring the sample just below the top of the Sun and into the column bed 6. Without disturbing the sand or alumina, carefully fill the column with hexane and elute the waste beaker, Change the receiver to your first labeled beaker (Fraction #1) When the first colored ban is about 1 cm from the bottom of the column 7. Change the eluting solvent to a 1:1 hexane/TBME. Fill the columns with a mixed solvent elute most of the first band 8. Change the eluting solvent to pure TBME. Collect this intermediate fraction in a labeled beaker (Fraction #2). When the second be your color band is about 1 cm from the bottom of the column, place a labeled beaker (Fraction #3) 9. Change the eluting solvent to acetone. Elute the entire second band with acetone (Fraction #3) 10. Run A TLC-applying five spots: Fraction #1, Fraction #2, the content of the waste beaker, and the samples of each of the pure compounds. Analyze the results of your separation by column chromatography by analyzing what is being viewed on the TLC plate. Part 2: Separation of a Mixture of Nitroanilines Isomers 1. Prepare the column as described 2. Use 7in of alumina 3. Weigh out 0.05g each of orthonitroaniline and para-nitroaniline and dissolve them together in a ½ mL of acetone. Crush the solid with the tip of the glass rod and grind the solid so goes through Use a pipet when putting the liquid into the column Continuously adding the solute and be careful when doing it To move the Acetylferrocene from the top a more polar solvent is added which is hexane/TBME Make sure to collect the middle layer (waste) which tends to be a mixture of Acetylferrocene and Ferrocene Add more acetone so that the Acetylferrocene will move down After separating all of the mixtures spot them do TLC Rf value results: Rf value(1): 0.89 Rf value(2a): 0.88 Rf value (2b): 0.40 Rf value(3): 0.40 Rf value(4): 0.88 Rf value(5): 0.39 Follow the same methods as part 1 of the experiment they can dissolve more readily 4. Run the solvent to just below the top of the sand on the column, and carefully apply the solution of nitroanilines to the column with a Pasteur pipet 5. Drain a small amount of solvent to bring the sample just below the top of the sand and onto the alumina bed. 6. Without disturbing the sand or alumina, carefully fill the column with hexane and elute the beaker. When enough hexane is present the band of yellow will steadily begin to move down the column and will become stationary and the polarity of the eluting solvent must be increased 7. Run hexane to the top of the sand and switch to a 9:1 hexane/acetone mixture 8. When The first yellow reaches the top of the glass wall plug, begin to collect the eluant in a flask labeled “Fraction #1”. Collect until essentially all of the first band of color has been removed and then switch to a 5:1 hexane/acetone solvent system 9. As soon as the first band of color is removed, run the solvent down to the top of the sand and switch to pure acetone. Meanwhile, collect the solvent that elutes between the twocolored bands into a fresh Beaker, and save it. Switch to a flask labeled “Fraction #2” as soon as the second band of color reaches the top of the glass wool, or when the eluant becomes a deep yellow again. 10. Run A TLC-applying five spots: Fraction #1, Fraction #2, the content of the waste beaker, and the samples of each of the pure compounds. Analyze the results of your separation by column chromatography by analyzing what is being viewed on the TLC plate. Calculations: Part 1: TLC Analysis of a Mixture of Colored Compounds 1) Fraction #1 Rf value(1): 11.6 cm/13cm = 0.89 2) Fraction #2 Rf value(2a): 11.4 cm/13cm = 0.88 3) Fraction #3 Rf value (3): 5.2 cm/13cm = 0.40 4) Pure Ferrocene Rf value(4): 11.5 cm/13cm = 0.88 Rf value(2b): 5.2 cm/13cm = 0.40 5) Pure Acetylferrocene Rf value(5): 5.1 cm/13cm = 0.39 Conclusions: In this lab column chromatography was used for this experiment and the purpose for that was to separate and purify solids and liquids. The video that was played in the lab had gone over the experiment and shown the process of performing column chromatography. For the first part of the lab Ferrocene and Acetylferrocene were being tested. When performing the experiment Ferrocene had moved a lot faster down the column since it is nonpolar compared to Acetylferrocene which had taken a lot longer since it polar. Hexane was the solvent that was used to move the compounds down the column and then t-butyl methyl ether also had to make the solvent more polar to have Acetylferrocene move down the column. Five different samples were collected from the Column Chromatography; 1)Fraction #1, 2)Fraction #2, 3)Fraction #3, 4)Pure Ferrocene, and 5)Pure Acetylferrocene; which then were tested with TLC to find the Rf values. Based on the Rf values the results can be determined if the samples are polar, slightly polar, or nonpolar. Sample 1)Fraction #1 had a Rf value(1) of 0.89 meaning that this sample is very nonpolar. For sample 2)Fraction #2 has two different Rf values, Rf value(2a) equaled to 0.88 which is very nonpolar and Rf value(2b) equaled to 0.40 which is a slightly polar compound. For sample 3)Fraction #3 had a Rf value(3) of 0.40 which is a slightly polar compound. For sample 4)Pure Ferrocene had a Rf value(4) of 0.88 which is very nonpolar. For sample 5)Pure Acetylferrocene had a Rf value(5) of 0.39 which is slightly polar. Based on all of the information gained from calculations Fraction #1 is almost identical to Pure Ferrocene but might be mixed with another compound which had changed its Rf value. Fractions #3 is almost identical to Pure Acetylferrocene but might be mixed with another compound which had changed its Rf value. Fraction #2 is a mixture of Pure Ferrocene and Pure Acetylferrocene based off of the data from the Rf value. Fraction #3 most likely is the waste layer between Pure Ferrocene and Pure Acetylferrocene. For the second portion of the experiment the Nitroaniline isomers; Ortho-nitroaniline & Para-nitroaniline were tested. There were three different samples being tested with TLC after going through Column Chromatography; 1) Pure Ortho-nitroaniline 2) Middle Layer (Waste) and 3)Pure Para-nitroaniline. Based off of the TLC 1) Pure Ortho-nitroaniline is slightly polar. For sample 2a) Middle Layer (Waste), it is slightly polar meanwhile 2b) Middle Layer (Waste) was polar. The last sample on the plate 3)Pure Para-nitroaniline is polar. Based on this information the Middle Layer (Waste) is a mixture of Pure Ortho-nitroaniline and Pure Para-nitroaniline. Errors that can occur in this lab could be overfilling the column with too much of the solvent. This could alter the results intended or even have the solvents spilling outside of the column. To prevent this from happening, use a pipet and put a few drops at a time instead of squeezing it all out at once. Another possible source of error is not changing the flask after collecting each fraction or pure substance. This can alter the results and would lead to redoing the experiment to obtain the correct results. To avoid this, make sure to have multiple collecting flask and paying attention to the column at all times to make sure that the stopcock is closed when placing a new collection flask. Post-Lab Questions: 1. The reason why it is important to apply the sample to the column in the smallest amount of solvent possible is because too much of the solvent we'll make the sample too diluted and will not lead to a proper separation between the components. 2. The purpose of the sand on top of the column is to be a layer between the solvent and the stationary phase. The purpose of the sand on the bottom of the column is to make sure that none of the stationary phase will be able to pass through the bottom of the column. 3. The melting point determination and TLC serve as important methods for finding impurities that are possibly present in the solid sample. Although the melting point determination does allow for finding impurities, the melting point range will change even if there is a small amount of impurities present in the solid sample. On the other hand, TLC displays the total amount of impurities present during separation. 4. Based on the information given in this question, the solvent which is being utilized is hexane and is a nonpolar compound. The reason for the orange compound not moving is because it’s polar and fond of the stationary phase that is also polar. To make this experiment work correctly, instead of using hexane, use a solvent that is polar. This will result in the orange compound to move down with the solvent. 5. Based on the information given in this question, dichloromethane is the solvent being used and is polar. This is what is causing the sample from moving. To make this experiment work correctly the solvent has to be changed to a less polar compound and then this will lead to the separation needed for the experiment.