Objective Statement: In this lab, I will learn about the procedure of column chromatography to purify compounds. Learning how to prepare and understand column chromatography to produce fractions of purified compounds for analysis by mass spec.
Embedded Files
Ferrocene
Polarity: non-polar
Mass: 186.04 g/mol
MF: C10H10Fe
Acetylferrocene
Polarity: slightly polar
Mass: 228.07 g/mol
MF: [Fe(C5H4COCH3)(C5H5)
Hexane
Polarity: non-polar
Mass: 86.18 g/mol
MF: C6H14
3-Ehtylhexane
Polarity: slightly polar
Mass: 114 g/mol
MF: C8H18
Pre-Lab Questions:
In order from the bottom to the top, list the layers in your column. You can answer by following this template: First I will put in xxx and then yyyy and then….
-First I will compact the column with silica gel, then put a mixture of ferrocene and acetylferrocene on top of that, with the solvents being on the highest layer.
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?
-Add sample to column
-Add hexane on top of the sample through the column
-Add ethyl to make an ethyl/hexane mixture to remove the other components
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 a material quality that describes how readily a substance vaporizes. Where substances with high volatility need high energy and harsh environments to cause vaporization, gas chromatography introduced the variables needed to reach vaporization. Where liquid chromatography has almost no variables to induce vaporization so it is what low-voltage compounds go through for purification.
4. What force pulls the mobile phase through the stationary phase? Your answer can be a single word.
-Capillary action (polarity)
5. Which is the more polar solvent system? The 100% hexane or 50% hexane/50% ether. Explain.
-Hexane is a straight-chain alkane meaning it is not polar ever. Where the half and half ethyl: hexane mix is slightly polar due to the ethyl branches on hexane. This means that the ethyl: hexane is the more polar solvent system.
6. If you were carrying out a chromatographic resolution of 0.5 g how much silica is recommended to use?
-7.5g of silica (Book states 15.0g of silica for 1.0g of sample).
7. What is “channeling” concerning column chromatography?
-"Channeling" is uneven flow within the chromatography column due to improper packing.
8. For the sake of time, you will change solvent abruptly. What can you expect to occur and why does this occur?
-A less-polar solvent is first used to elute a less-polar compound. Once the less-polar compound is out of the column, a more-polar solvent is added to elute the more-polar compound.
9. What is the optimal flow rate for column chromatography?
-Around 1-2 mL/min.
10. What should be the elution order? Will ferrocene elute first or acetyl ferrocene? Explain.
-Ferrocene will elute first. When using a non-polar mobile phase of hexane first, the non-polar compound, ferrocene will elute out of the column first. Followed by the elution of acetylferrocene after the introduction of the slightly-polar ethylhexane solvent system.
Procedures:
acquired a column chromatography chamber and placed a small ball of cotton at the bottom of the column with around 1.0 cm of sand above the cotton to protect the cotton from the gel.
In a separate column, measured out 10.0 cm of dry silica gel and poured it into a separate Erlenmeyer flask with enough hexane to make the mixture into a slurry. Want it to be a very light slurry, like an almost completely melted slushy.
Added the wet silica gel to the chromatography column with the bottom open to allow for hexane to drip out of the column. Used the excess hexane dripping from the column to rinse out the Erhlenmeyer flask and the top of the column to ensure all the silica gets added to its layer in the chromatography column. The hexane dries extremely fast causing some of the silica to revert into a powder form and not be able to move to the bottom of the column.
Made a 1cm layer of sand on top of the silica gel
Slowly poured .50 g of ferrocene mixture on top of the sand layer and used a small amount of hexane out of a pipette to wash any ferrocene stuck to the sides of the column.
After the hexane runs through the ferrocene mixture, add another 2cm layer of sand on top of the ferrocene to create a protection layer for the solute layer.
Added 50 mL of hexane on top of the final sand layer and let the hexane drip out the bottom of the column for reuse.
After about 40 mL of the first hexane wash went through, the first compound band was near the bottom of the column. A small column appeared towards the bottom of the channel because we dropped a mixing stick in the gel earlier in the setup.
Added around 40 mL of ether to the removed 40 mL of hexane to create a 1:1 hexane/ether solution. This will help to bring the second, polar, compound ring to the bottom of the column for extraction.
Took samples every 2 minutes from the bottom ferrocene layer (orange) starting to drip to the top acetylferrocene (red) layer went through the bottom of the chromatography column. The excess solvent cannot be reused after the point of the first layer extraction since the solvent has the pressure of ferrocene. Add 1:1 solvent mixture as needed to not allow the column to dry out.
Every other sample collection was analyzed by TLC for purity. See results below
Results:
-10 fractions collected (5 in ferrocene ring and 5 in acetyl ferrocene layer).
-Fractions were collected every other 2 minutes from the start of the first ring to the end of the second ring. This was to not collect constant fractions and only analyze every other fraction.
-The ferrocene fractions were very light orange possibly yellow while the acetyl fractions were much darker orange, and the first two fractions of the acetyl were lighter compared to the other acetyl fractions possibly due to the presence of some ferrocene left over in the column.
Ferrocene TLC
Ferrocene TLC
Solvent System: 1:10 ethyl acetate/hexane
The first spot is the Standard at an RF 4.0 cm /4.5 cm = .89
Procedure sample at an Rf = 3.95 cm /4.5 cm = .88
The spots were mainly yellow, a bit orange.
Acetlyferrocene TLC
Acetlyferrocene TLC
Solvent System: 1:10 ethyl acetate/hexane
The first spot is the standard at an RF = .75 cm /3.5 cm = .21
Procedure sample at an RF = .7 cm - .9 cm /3.5 cm = .2 - .25
The spots were a dark orange almost red-orange.
Conclusions:
The purpose of this lab was to use column chromatography with a two-part solvent system to separate and determine the purity of ferrocene and acetylferrocene. The two-part solvent system was used to separate the ferrocene and acetyl ferrocene in the mixture by first using a 95:5 hexane/ether solvent mixture to extract the non-polar ferrocene. As the solvent was mainly non-polar, the ferrocene moved down the column first and the acetyl ferrocene was left behind. This helped to create fractions of pure ferrocene before the acetyl ferreocene left the chamber. Once the Perricone ring (orange in the picture above) was near the bottom, a 50:50 hexane/ether mixture was added to the chamber to bring the remaining acetate out of the column. By splitting up the non-polar and slightly polar solvents, the compounds, varying slightly in polarity, were able to be extracted separately. We can see that this did happen as almost all of our fractions from the first orange ferrocene ring were nearly completely pure when compared to the RF value of the standard. However, the acetyl fractions varied slightly and that could mainly be due to the presence of some ferrocene being left behind at the end of the first compound ring transitiong into the second. Overall, the TLC analysis shows that the spots only had one dot, sharing similar Rf values, indicating pure and effecting purification. Indicating that column chromatography is an effective procedure for the purification of acetyl ferrocene from ferrocene.
Reflections:
This lab helped to teach the technique of packing a column chromatography chamber. Along with understanding the importance of knowing what the polarity of your desired compound is, and what solvents have similar polarities to solute and extract your desired compounds. The compounds with stronger attraction to the stationary phase will move slower through the column when compared to compounds with less attraction to the stationary phase. In this lab, the stationary phase was silica gel, where the more polar acetly ferrocenne had less attraction and did not move effectively until the introduction of ether in the solvent mixture moved it through the chamber. TLC was then used to determine the purity of the fractions by comparing every other fraction to a standard to see how the purity of the ferrocene to acetly ferrocene changed over the procedure. The only major hiccup we experienced during this lab was that we accidentally dropped a mixing stick into our column while we were packing it so we did see a little channeling towards the bottom of the chamber due to it disturbing the sand barrier between the cotton at the bottom. For the next lab, we will make sure to be more careful during the preparation of our procedure as it is just as important to the results as effectively doing the procedure techniques. I would also like to use alumina in the column to see how that affects the effectiveness of column chromatography.
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 is slightly acidic and it often retains basic compounds. The opposite is true for Alumina, which is slightly basic and will not allow acidic compounds to solute through it. Alumina also is good for weak acids due to its polarity.
2. The kind of chromatographic separation that you are doing is called the normal phase. How is the “reversed phase” different?
-Reversed-phase is different because it is controlled by polarity changes of the solvent. This will act as the mobile phase of the chromatography chamber and make the stationary phase the non-polar phase.
3. Briefly explain flash chromatography and how it is different from gravity chromatography.
-Flash chromatography is applied for fast partitions of purification. Done by the use of air pressure to push the compounds through the chambers at an accelerated pace using smaller silica particles for finner filtration.
4. Briefly explain HPLC and how it is different from gravity chromatography.
The difference is in the pressure of HPLC. The pressure used during HPLC is extremely higher than gravity column chromatography for rapid separation or ocmpounds that will take longer to go through the chamber due to very slight polarity. Everything is also automated since it is an instrument.
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