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Objective: The purpose of this lab is to gain an understanding of the process of column chromatography. Column chromatography allows for enatiomers of a racemic mixture to be seperated from each other. The purity of this seperation is then tested using TLC.
Compounds of Study:
Ferrocene
Ferrocene
Formula: (C10H10Fe ),
Molar Mass: (186.04 g/mol)
MP: (172.5°C )
Polarity: (Non-Polar)
Image from Sigma-Aldrich
Acetyl Ferrocene
Acetyl Ferrocene
Formula: (Fe(C5H4COCH3)(C5H5))
Molar Mass: (228.07 g/mol)
MP: (81-83°C )
Polarity: (Polar)
Image from Sigma-Aldrich
Pre-Lab:
1. In order from the bottom to the top, list the layers that will be in your column. You can answer by following this template: First I will put in xxx and then yyyy and then….
First, a "slurry" that is composed of hexane and silica gel is added to the column. Then additional hexane is added to the column. After the hexane has been added, ferrocene is added to the column. More hexane is then added to get any resin off of the sides of the column and allows for the greatest collection of material to occur.
2. Generally outline what you will do in the lab. First, you will prepare your column and then what will you do and then what comes next?
Once the column has been prepared the sample will be loaded into the column. Ferrocene and additional hexane will be added to the column before it is allowed to run until 100 mL has run through the column. At this point, ether will be added to create a 10% ether/hexane mixture and then the solvent will be changed to a 50% ether/hexane mixture when one colored solvent reaches the end of the column. As the column runs, a fractional analysis will be conducted for every other 2 mL collected. Fractional analysis is conducted by TLC with the pure substance for comparison and determining their Rf values.
3. The text states that liquid chromatography is used to purify substances that have low volatility and that gas chromatography is used to purify substances that have high volatility. What does this statement mean? Your answer must include a definition of volatility.
Volatility is defined as the tendency of a substance to vaporize at a given temperature. Gas chromatography is used to purify substances with high volatility because they can't be purified using liquid chromatography due to how easily they will vaporize.
4. What force pulls the mobile phase through the stationary phase? Your answer can be a single word.
Polarity.
5. Which is the more polar solvent system? The 100% hexane or 50% hexane/50% ether. Explain.
The 50% hexane/50% ether solution is more polar. Ether is slightly polar, while hexane is not, this is due to the C-O bond found within ether which makes the 50:50 solution more polar than the pure hexane solution.
6. If you were carrying out a chromatographic resolution of 0.5 g how much silica is recommended that you use?
7.5 grams of silica.
7. What is “channeling” with respect to column chromatography?
Channeling occurs when a solvent flows unevenly through the column. This is caused by improper packing of the column.
8. For the sake of time, you will be changing solvent abruptly. What can you expect to occur and why does this occur?
It is possible to see a poor separation between substances within the column. It is also possible to see air bubbles appear in the column, caused by the changing of the solvent. Both of these issues may affect the final purity of the compound as enantiomers may not fully separate.
9. What is the optimal flow rate for column chromatography?
1-2 mL/min.
10. What should be the elution order? Will ferrocene elute first or acetyl ferrocene? Explain.
The Ferrocene will elut first. Ferrocene will elute first because it is the most non-polar compound present within the system, the non-polar compound will bind less tightly meaning it fully exits the column before the polar compound (acetyl ferrocene).
Column Chromatography
Experimental:
Started by preparing the column for liquid chromatography, measured 15.00 cm of silica gel, and created a slurry with hexane.
Silica gel and slurry were loaded into the column. The silica was well packed, compact, and flat with no obvious channels seen.
"Packed" the column by adding 15.00 ml of hexane and allowing it to run through.
Hexane was drained into a 150.00 ml Erlynmyer flask.
Hexane readded to the column and above steps repeated.
0.50 g of ferrocene was measured.
Two layers of sand were added to the column with the ferrocene added in between them (The first layer of sand is the thin layer and the top layer of sand is the thick layer).
Slowly added 50.00 ml of Hexane and began to run the column.
Recycled hexane until the yellow band was near the bottom (when the yellow-colored band first started moving it was narrow but then became wider as it progressed through the column). As the column ran, the column became more yellow in appearance before becoming clear again.
Collected 12 samples of the yellow band.
Slowly added 50.00 ml of 95:5 hexane/ether mixture, and slowly added another 50 ml before the red sample could be collected. As the column ran, the column became a deep orange-red in appearance before becoming clear again (similar to the yellow-band, when the orange-colored band first started moving it was narrow but then became wider as it progressed through the column)
Collected 12 samples of the deep orange-red band.
Performed TLC using the yellow band (6, every other sample collected) samples and its standard to calculate RF.
Performed TLC using the deep orange-red band (6 for every other sample collected) samples and its standard to calculate RF as additional conformation for the experiment.
Results:
Column shortly after the ferrocene and hexane had been added.
Apperance of the yellow band (seen at the bottom of the column) and the orange band (seen at the top of the column) after both hexane and hexane/ether had been added to the column.
Appearance of the column toward the end of our experiment and as we began to collect the orange band samples.
Column Chromatography:
Twelve yellow-colored samples were obtained after the first 50.00 ml of hexane was added following the loading of ferrocene into the column.
Twelve orange-colored samples were obtained after 50.00 ml of 95:5 hexane/ether mixture was added to the column. The samples began deeper in color and then became lighter in color.
Pictured on the left is the column shortly after the ferrocene and hexane had been added to the column.
TLC:
Solvent 1:10 Ethyl Acetate/Hexane
TLC of Ferrocne
Pure Sample indicated by "P"
Solvent front = 3.5cm
Spot P = 3.0cm
Lab isolated 1st band of column chromatography = 3.0cm
Rf of Spot P = (3.0)/(3.5) = 0.86
Rf of Original Spot = (3.0)/(3.5) = 0.86
TLC of Accetlyferrocene
Pure Sample Indicated by "P AF"
Discussion: The TLC analysis indicates that the yellow band (first band) removed from the column was ferrocene. The Rf value of our lab-obtained samples matched that of the standard (Lab obtained Rf Value: 0.86, Pure Sample Rf Value: 0.86). The color of the TLC for the pure sample and the lab sample was also similar, providing further evidence that the compounds are the same.
My lab partner and I double-checked our work by creating a second TLC plate of the orange-red band (second band) which indicated that the substance removed from the column was acetyl-ferrocene based on its Rf values and the color of the control and experimental sports being similar in appearance.
These results are consistent with our predictions for this lab as we would expect ferrocene to be extracted first as it is more polar in nature than acetyl-ferrocene.
Conclusion: The compounds were effectively collected by column chromatography. This is indicated by the compounds behaving similarly to the standards used to identify them using TLC. Ferrocene was isolated first with the non-polar elute and the acetyl ferrocene was isolated second when the eluant became more polar.
Reflection: In this lab, I learned how a compound can be extracted from another using column chromatography. I gained a better understanding of how to identify polar and non-polar compounds in a column. By completing the post-lab, I was also able to learn more about other chromatography techniques. In this experiment, I practiced appropriate column packing techniques, such as loading silica in a way that allows it to lay flat and compact without channels and collecting specimens that can be used to assess the purity of the separation performed by column chromatography. One thing I would do differently if I were to repeat this lab is attempt to perform column chromatography using a different chemical to allow me to continue to practice and perfect my column chromatography skills using different compounds.
Post-Lab Questions:
1. Both Silica Gel and Alumina are common column chromatography resins. What is the chemical makeup of these resins and how would you choose between using one over the other?
Silica gel (SiO2) is suitable for polar and moderately polar compounds, while Alumina (Al2O3) is better for less polar compounds. When choosing which resin to use it is helpful to use, it is helpful to take into account the polarity of the compound.
2. The kind of chromatographic separation that you are doing is called the normal phase. How is the “reversed phase” different?
"Normal phase" is when the polar stationary phase separates compounds based on polarity using nonpolar solvents. In "reversed phase" chromatography, the nonpolar stationary phase separates compounds based on hydrophobicity using polar solvents. In reversed-phased chromatography, the resin is non-polar, causing non-polar compounds to stick and polar compounds to elute quickly which is the opposite of normal-phase chromatography.
3. Briefly explain flash chromatography and how it is different from gravity chromatography.
Flash chromatography is known for its rapid speed and ability to process relatively large sample volumes compared to traditional column chromatography. Flash chromatography is performed through the use of air pressure and silica particles that are smaller than those used in gravity chromatography.
4. Briefly explain HPLC and how it is different from gravity chromatography.
HPLC differs from gravity chromatography because HPLC operates at significantly higher pressures than gravity-based chromatography. HPLC is a fully automated procedure and is performed by an instrument while gravity chromatography is not.
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