Chromatography: Polarity Of Compounds
Chromatography
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•
•
Separation based on polarity of compounds
Two potential phases for a compound to exist in: mobile and stationary
Partitioning of compounds between mobile phase and stationary phase occurs: o Compounds that are less polar move more in the mobile phase, those that are more polar “stick” more on the stationary phase o These polarity differences cause compounds move at different rates and therefore can be separated
1. Mobile Phase: the phase the moves; can be gas or liquid (solvent; eluent)
• For liquids, the polarity of a solvent is defined as its ability to dissolve polar organic compounds.
• Compounds dissolved into the solvent spend more time moving in the mobile phase • Non-polar solvents can only dissolve …show more content…
non-polar compounds.
• As the relative polarity for solvents increases, the solvents will be able to dissolve more compounds, beginning with the non-polar ones and then increasing numbers of more polar ones.
• Very polar solvents will be able to dissolve ALL compounds.
For Chromatography: “Polar Dissolves More” not “like dissolves like”
Some Common Solvents: (listed by increasing polarity)
• Petroleum ether (C5 isomer compounds, like pentane; not a true “ether”)
• Ligroin (C6 isomer compounds, such as hexane)
• Diethyl Ether (CH3CH2OCH2CH3)
• Dichloromethane (CH2Cl2)
• Ethyl Acetate (ester: CH3CH2OC(O)CH3)
• Methanol (alcohol: CH3OH)
• Acetic Acid (carboxylic acid: CH3CO2H)
And of course – mixtures of the above solvents can be used.
Consider the following example: Compound A is a non-polar compound and compound
B is a polar compound.
Add Petroleum ether (non-polar!) – Which will dissolve?
Only Non-Polar A
Add Diethyl Ether (more polar!) – Which will dissolve?
Both!
1
In today’s lab, we’ll begin the separation for column chromatography with petroleum ether and only remove the non-polar compound. We will then switch to a more polar mixture of petroleum ether and diethyl ether to remove a more polar …show more content…
compound.
If we used the more polar mixture first, what problem would occur?
Both compounds would travel down the column at the same rate because both are dissolved in the more polar mixture. No separation!
2. Stationary Phase – Alumina or Silica on a solid support
• The more polar the compound, the stronger the compound adheres (“sticks”) to the adsorbent.
• Silica Gel (SiO2) and Alumina (Al2O3) are the two most common adsorbents for separations in the organic lab.
Functional Groups (listed by increasing polarity):
Alkanes
Alkenes, alkynes and aromatics
Ethers (R-O-R)
Esters (RCO2R
Ketones and aldehydes (RC(O)R or RC(O)H)
Amines (:NR3)
Alcohols (ROH)
Carboxylic Acids (RCO2H)
Partitioning Effect:
Generally, polar compounds spend more time on the stationary phase (“stuck”) and non-polar compounds spend more time in the mobile phase (moving in the solvent).
The goal is to use a solvent system that only moves the least polar compound, until it is completely removed from the system. Then a more polar solvent can be added to move the next compound.
Column Chromatography:
•
•
Used for purification of organic compounds (solids or liquids)
Size of separation can range from milligrams to kilograms
2
Sand
alumina or silica gel
Sand
Packing of the Column: -demo• Must remove air bubbles – pockets of air allow compounds to travel in the mobile phase faster through air pockets resulting in separation that is not uniform • Column must be assembled in a vertical fashion, so bands will travel downwards in an even and uniform fashion for the best separation
•
Crooked bands tend to stay overlapped slightly
•
Sample must be loaded in the most concentrated method in order to start with the narrowest band possible for best separation
3
o wide bands tend to stay wide (or get wider) and overlap
•
•
•
•
May use a single solvent or may change solvents during column
Use gravity or gas pressure to move the mobile phase through the stationary phase (“flash column chromatography”)
Collect fractions containing the bands and evaporate the solvent.
Watch for any crystals “growing” on the bottom of the column - these should be rinsed into the appropriate vial.
Thin-Layer Chromatography (TLC):
•
•
•
Used to identify compounds (like GC)
Used to check the purity of a compound
Used to check a reaction’s progress
Only need micrograms of material to do this process.
Major difference between column and TLC is the direction of the flow of the mobile phase!
Similarities:
• Mobile Phase: Same Solvents
• Stationary Phase: Same Alumina or Silica applied so some solid support
• Same partitioning effect based on compound polarity
4
x
Developing Chamber and Cover
-solvent in bottom
-add a "wick"
Microcapillary Tube
x
x
TLC Plate, covered with
Al2O3 or SiO2
Process Summary: -demo1. Spotting the Plate (application of the compounds)
• TLC plate is marked with a pencil (not pen) to show point of origin (where compound is to be applied). This point of origin must be about 1 cm above the bottom of the plate (above the level of the solvent used as the mobile phase).
This avoids having the mobile phase wash the compounds off when the plate is lowered into the developing chamber.
• The microcapillary tube uses capillary action to pull liquid into tube.
• Compound is released by capillary action upon gentle tapping onto the adsorbent.
2.
•
•
•
•
Developing the Plate (movement of eluent through adsorbent)
Developing Chamber – glass container, such as beaker or jar, with a cover.
Mobile phase (eluent) uses capillary action to “climb” the plate in a vertical fashio. You must have a “wick” inside the chamber to provide a saturated atmosphere so the eluent does not evaporate off the plate as it climbs up the plate. The plate must not touch the wick or side of chamber when it is being developed. This would cause the mobile phase to move in a sideways or diagonal fashion also, thus ruining the “lanes” of travel by the compounds and preventing any identification.
Plate must be monitored. You can see how high the eluent has moved as the eluent slowly saturates the plate as capillary action moves it upwards. The front edge of this movement is called the “solvent front”. The plate must be removed prior to the eluent reaching the end of the plate. You need the
5
measurement for the solvent front to do future calculations. If the solvent front has reached the end, you have no idea how far the spot(s) have moved relative to the distance the solvent has travelled.
3. Visualization
• Typically organic compounds are colorless and not seen by the human eye.
• To visualize, you may use iodine, a UV lamp, or one of many organic stains. Circle the spot with a pencil. Record the appearance of the spot on the plate.
• In today’s experiment, ferrocene and acetylferrocene are colored compounds.
4. Calculations
Rf value: the distance the spot travels divided by the distance the solvent travels
(from the origin to the solvent front). This value will be between 0 and 1 and is not expressed as a percentage. All measurements done in metric units (cm or mm)
Rf values are commonly used for identification purposes and are cited with the appropriate solvent system used to attain the Rf value.
• the same compound with always have the same Rf value under the same conditions (i.e.
same solvent system). To identify an unknown compound, spot known compounds onto the same plate as your unknown compound and run the compounds side-by-side to compare.
• The spots for the same compounds will not only have the same Rf value but also have the same appearance.
Same Compound or Not?
TLC can also be used to check the purity of a compound. A pure compound will always appear as a single spot (because it is a single compound) regardless of what solvent system is used for developing the plate. The solvent system should be neither very polar nor very non-polar when checking this. This should result in an
Rf value in the range of 0.2-0.8, ideally. Non-polar solvents would not move the spot enough and polar solvents would move them too much to be able to evaluate.
Pure or Not?
6
non-polar solvent polar solvent Using TLC to follow the progress of a reaction:
In the following reaction, the alcohol product is more polar than the starting ketone. With regard to the TLC, you would expect that the ketone appear higher on the TLC plate (higher Rf value) while the alcohol would appear lower on the plate
(lower Rf
value).
O
OH
LiAlH4
The starting compound is spotted on a TLC plate with the reaction mixture, at timed intervals to monitor the progress. The reaction SHOULD show a decrease in the starting material and an increase in a new MORE POLAR compound spot.
x sm x rxn T = 0 min
x sm x rxn T = 15 min
x sm x rxn T = 30 min
x sm x rxn T = 45 min
x sm x rxn T = 60 min
7
•
•
•
Separation based on polarity of compounds
Two potential phases for a compound to exist in: mobile and stationary
Partitioning of compounds between mobile phase and stationary phase occurs: o Compounds that are less polar move more in the mobile phase, those that are more polar “stick” more on the stationary phase o These polarity differences cause compounds move at different rates and therefore can be separated
1. Mobile Phase: the phase the moves; can be gas or liquid (solvent; eluent)
• For liquids, the polarity of a solvent is defined as its ability to dissolve polar organic compounds.
• Compounds dissolved into the solvent spend more time moving in the mobile phase • Non-polar solvents can only dissolve …show more content…
non-polar compounds.
• As the relative polarity for solvents increases, the solvents will be able to dissolve more compounds, beginning with the non-polar ones and then increasing numbers of more polar ones.
• Very polar solvents will be able to dissolve ALL compounds.
For Chromatography: “Polar Dissolves More” not “like dissolves like”
Some Common Solvents: (listed by increasing polarity)
• Petroleum ether (C5 isomer compounds, like pentane; not a true “ether”)
• Ligroin (C6 isomer compounds, such as hexane)
• Diethyl Ether (CH3CH2OCH2CH3)
• Dichloromethane (CH2Cl2)
• Ethyl Acetate (ester: CH3CH2OC(O)CH3)
• Methanol (alcohol: CH3OH)
• Acetic Acid (carboxylic acid: CH3CO2H)
And of course – mixtures of the above solvents can be used.
Consider the following example: Compound A is a non-polar compound and compound
B is a polar compound.
Add Petroleum ether (non-polar!) – Which will dissolve?
Only Non-Polar A
Add Diethyl Ether (more polar!) – Which will dissolve?
Both!
1
In today’s lab, we’ll begin the separation for column chromatography with petroleum ether and only remove the non-polar compound. We will then switch to a more polar mixture of petroleum ether and diethyl ether to remove a more polar …show more content…
compound.
If we used the more polar mixture first, what problem would occur?
Both compounds would travel down the column at the same rate because both are dissolved in the more polar mixture. No separation!
2. Stationary Phase – Alumina or Silica on a solid support
• The more polar the compound, the stronger the compound adheres (“sticks”) to the adsorbent.
• Silica Gel (SiO2) and Alumina (Al2O3) are the two most common adsorbents for separations in the organic lab.
Functional Groups (listed by increasing polarity):
Alkanes
Alkenes, alkynes and aromatics
Ethers (R-O-R)
Esters (RCO2R
Ketones and aldehydes (RC(O)R or RC(O)H)
Amines (:NR3)
Alcohols (ROH)
Carboxylic Acids (RCO2H)
Partitioning Effect:
Generally, polar compounds spend more time on the stationary phase (“stuck”) and non-polar compounds spend more time in the mobile phase (moving in the solvent).
The goal is to use a solvent system that only moves the least polar compound, until it is completely removed from the system. Then a more polar solvent can be added to move the next compound.
Column Chromatography:
•
•
Used for purification of organic compounds (solids or liquids)
Size of separation can range from milligrams to kilograms
2
Sand
alumina or silica gel
Sand
Packing of the Column: -demo• Must remove air bubbles – pockets of air allow compounds to travel in the mobile phase faster through air pockets resulting in separation that is not uniform • Column must be assembled in a vertical fashion, so bands will travel downwards in an even and uniform fashion for the best separation
•
Crooked bands tend to stay overlapped slightly
•
Sample must be loaded in the most concentrated method in order to start with the narrowest band possible for best separation
3
o wide bands tend to stay wide (or get wider) and overlap
•
•
•
•
May use a single solvent or may change solvents during column
Use gravity or gas pressure to move the mobile phase through the stationary phase (“flash column chromatography”)
Collect fractions containing the bands and evaporate the solvent.
Watch for any crystals “growing” on the bottom of the column - these should be rinsed into the appropriate vial.
Thin-Layer Chromatography (TLC):
•
•
•
Used to identify compounds (like GC)
Used to check the purity of a compound
Used to check a reaction’s progress
Only need micrograms of material to do this process.
Major difference between column and TLC is the direction of the flow of the mobile phase!
Similarities:
• Mobile Phase: Same Solvents
• Stationary Phase: Same Alumina or Silica applied so some solid support
• Same partitioning effect based on compound polarity
4
x
Developing Chamber and Cover
-solvent in bottom
-add a "wick"
Microcapillary Tube
x
x
TLC Plate, covered with
Al2O3 or SiO2
Process Summary: -demo1. Spotting the Plate (application of the compounds)
• TLC plate is marked with a pencil (not pen) to show point of origin (where compound is to be applied). This point of origin must be about 1 cm above the bottom of the plate (above the level of the solvent used as the mobile phase).
This avoids having the mobile phase wash the compounds off when the plate is lowered into the developing chamber.
• The microcapillary tube uses capillary action to pull liquid into tube.
• Compound is released by capillary action upon gentle tapping onto the adsorbent.
2.
•
•
•
•
Developing the Plate (movement of eluent through adsorbent)
Developing Chamber – glass container, such as beaker or jar, with a cover.
Mobile phase (eluent) uses capillary action to “climb” the plate in a vertical fashio. You must have a “wick” inside the chamber to provide a saturated atmosphere so the eluent does not evaporate off the plate as it climbs up the plate. The plate must not touch the wick or side of chamber when it is being developed. This would cause the mobile phase to move in a sideways or diagonal fashion also, thus ruining the “lanes” of travel by the compounds and preventing any identification.
Plate must be monitored. You can see how high the eluent has moved as the eluent slowly saturates the plate as capillary action moves it upwards. The front edge of this movement is called the “solvent front”. The plate must be removed prior to the eluent reaching the end of the plate. You need the
5
measurement for the solvent front to do future calculations. If the solvent front has reached the end, you have no idea how far the spot(s) have moved relative to the distance the solvent has travelled.
3. Visualization
• Typically organic compounds are colorless and not seen by the human eye.
• To visualize, you may use iodine, a UV lamp, or one of many organic stains. Circle the spot with a pencil. Record the appearance of the spot on the plate.
• In today’s experiment, ferrocene and acetylferrocene are colored compounds.
4. Calculations
Rf value: the distance the spot travels divided by the distance the solvent travels
(from the origin to the solvent front). This value will be between 0 and 1 and is not expressed as a percentage. All measurements done in metric units (cm or mm)
Rf values are commonly used for identification purposes and are cited with the appropriate solvent system used to attain the Rf value.
• the same compound with always have the same Rf value under the same conditions (i.e.
same solvent system). To identify an unknown compound, spot known compounds onto the same plate as your unknown compound and run the compounds side-by-side to compare.
• The spots for the same compounds will not only have the same Rf value but also have the same appearance.
Same Compound or Not?
TLC can also be used to check the purity of a compound. A pure compound will always appear as a single spot (because it is a single compound) regardless of what solvent system is used for developing the plate. The solvent system should be neither very polar nor very non-polar when checking this. This should result in an
Rf value in the range of 0.2-0.8, ideally. Non-polar solvents would not move the spot enough and polar solvents would move them too much to be able to evaluate.
Pure or Not?
6
non-polar solvent polar solvent Using TLC to follow the progress of a reaction:
In the following reaction, the alcohol product is more polar than the starting ketone. With regard to the TLC, you would expect that the ketone appear higher on the TLC plate (higher Rf value) while the alcohol would appear lower on the plate
(lower Rf
value).
O
OH
LiAlH4
The starting compound is spotted on a TLC plate with the reaction mixture, at timed intervals to monitor the progress. The reaction SHOULD show a decrease in the starting material and an increase in a new MORE POLAR compound spot.
x sm x rxn T = 0 min
x sm x rxn T = 15 min
x sm x rxn T = 30 min
x sm x rxn T = 45 min
x sm x rxn T = 60 min
7