How to Perform Thin Layer Chromatography
Cut a large stock TLC plate from the box into rectangles., Mark the TLC plate., Prepare your chromatography solvent., Prepare your solutions., Take a small amount of solution., Lightly press the tip of the pipette to the intersection of one of the...
Step-by-Step Guide
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Step 1: Cut a large stock TLC plate from the box into rectangles.
You will probably need a glass cutter unless the plates are already pre-scored.
There are two sides to the TLC plate.
A silica side and a glass side.
The glass side is just smooth reflective glass, and the silica side is a powdery white substance that will flake off if you disturb it.
So be careful when handling these plates so as to not ruin the silica gel.
Touching the silica with your fingers should be avoided! Measure the dimensions for your
1.5 x 4 cm plates, or larger if you thing you need more space, but this size is usually sufficient.
Then lightly score the glass side, not the silica side, with the your blade or glass cutter.
It should look like the picture above, with obvious cuts in the glass.
Now carefully pick up the plate and break it by bending it with your hands away from you.
Glass will easily break along a smooth edge if scored.
Be very careful, but you must use enough force to break the glass.
This may take some practice if you have never done it before. -
Step 2: Mark the TLC plate.
Draw a line in pencil about
0.5 cm from the bottom straight across.If you are doing a simple reaction, as in most undergraduate labs, draw three evenly spaced vertical dashes on the horizontal line.
Label these lines as starting material (SM), co-spot (C), and reaction (R).
This will be explained more in later steps.
Only use pencil to write on the TLC.
If you use pen or marker it will ruin your TLC plate because these media contain dyes which are organic compounds that will develop along with your compounds of interest.
Graphite will not move with the solvent.
Write very gently, otherwise you will scrape off the silica. , A classic solvent system is ethyl acetate and hexane because they mix easily and the polarity of the solvent is easily adjusted.
A good place to start is with 20% ethyl acetate in hexane, which is the same as a 1:4 solution of ethyl acetate to hexane.
You can easily measure out 1 mL of ethyl acetate and 4 mL of hexane in a graduated cylinder, or other liquid-measuring tool, and then combine them to give the desired solution.
In an enclosed jar, place some 1:4 ethyl acetate:hexane so that the bottom of the jar is just barely covered.
Close the lid on the jar, or else the solvent will quickly evaporate and you will have to add more. , Take the starting material for your reaction and dilute it in a small vial with ethyl acetate.
TLC is very sensitive and even a 1% solution of your compound is plenty.
You really only need a very dilute solution for this procedure.
In fact, if your solution is too concentrated, your spots will streak and you will get no useful information from your TLC plate.
If your reaction is also a nice solution you can spot from it directly, but if your reaction mixture is more complex you may have to remove a small amount and dilute it in a suitable solvent (such as ethyl acetate) to make a solution.The best solutions to spot are clear, but they don't have to be.
The color of the solution does not matter.
In the picture above, the solution is clear and colorless because this compound is not colored, but oftentimes the solutions have color to it. , Submerge the tip of a glass pipette into one of the reaction solutions you just made, and the pipette it will suck up a small amount.
This is called capillary action and many people are surprised that this works.
See the above picture for proof that this actually works.
Simply stick the end of the pipette into the liquid, and some of it will come into the pipette.
You do not need very much solution for spotting on the plate, so the tip of a pipette is plenty. , It is very important to spot lightly.
If you press too hard, you will deposit too much material, making big spots which will overlap, causing the TLC to be difficult to read.
This step will take practice, so you may want to try this several times, trying to get as small of spots as possible before continuing onto the next step.
Spot the starting material on the first and second dashes, and the reaction solution on the second and third dashes.
The first dash is your starting material reference (SM), the middle spot is a co-spot (C), and the third spot is the reaction reference (R).
This type of spotting makes the developed plate very easy to read.
The the above picture for visual clarification. , Simply set the plate down and wait a minute or so for the solvent to evaporate off the plate.
You cannot run the plate if there is still solvent.
You will be able to see a wet spot on the plate if there is solvent.
You need not wait long for such volatile solvents as ethyl acetate and hexane.
A minute is more than enough time for these solvents to evaporate. , Using tweezers, gently place the TLC plate into the solvent jar, with the spotted end of the plate at the bottom close to the solvent.
Submerge the bottom edge of the plate into the solvent, allowing the solvent to run up the plate, carrying the compounds along with it.
Cover the jar with the lid.
This will prevent solvent from evaporating and make the development stage much faster.
This is the stage where separation occurs.
So be patient and wait 2-3 minutes for the solvent to reach the top of the plate before you remove it.
Waiting longer usually provides better results, so long as you don't let the solvent flow past the upper edge. , Once the solvent comes close to reaching the upper edge of the plate, use tweezers to carefully remove it from the chamber.
Quickly mark in pencil the highest place the solvent reached.
This is called the solvent front and is important to take note of for later calculations.
Do not touch the silica with your fingers as you could smudge the spots or deposit compounds from your hands onto the plate, making your plate useless. , Again, wait for the solvent to evaporate off the plate.
This should only take a minute or less if you are using a volatile solvent such as ethyl acetate hexane, but it is important to not have solvent on your plate. , Place the plate under an ultraviolet light.
You should notice several spots at different locations, corresponding to different compounds.
The presence or absence of spots corresponds to the progress of a reaction, so information about a reaction can be easily determined in this way.
Use a pencil to outline the spots so you can continue to analyze the plate after you remove it from the UV source.
Look at the above picture and notice the different spots at different heights. , This is simply the ratio of how far the spots have moved to how far the solvent has moved.
Use a ruler to measure the distance to the solvent front and to the middle of the spots.
Divide the distance the spots moved by the distance the solvent moved.
This will give you a number between 0 and
1.
By calculating the Rf of known compounds, you can identify them on a TLC plate.
Different compounds will almost always have different Rf values, making them easy to distinguish. -
Step 3: Prepare your chromatography solvent.
-
Step 4: Prepare your solutions.
-
Step 5: Take a small amount of solution.
-
Step 6: Lightly press the tip of the pipette to the intersection of one of the dashed lines and the horizontal line you made previously on the silica side.This will cause the solution in the pipette to be sucked onto the silica
-
Step 7: resulting in a small circle of solvent and compound.
-
Step 8: Dry the TLC plate.
-
Step 9: Develop the plate.
-
Step 10: Remove the plate.
-
Step 11: Dry the plate.
-
Step 12: Visualize the plate.
-
Step 13: Calculate the retardation factor (Rf).
Detailed Guide
You will probably need a glass cutter unless the plates are already pre-scored.
There are two sides to the TLC plate.
A silica side and a glass side.
The glass side is just smooth reflective glass, and the silica side is a powdery white substance that will flake off if you disturb it.
So be careful when handling these plates so as to not ruin the silica gel.
Touching the silica with your fingers should be avoided! Measure the dimensions for your
1.5 x 4 cm plates, or larger if you thing you need more space, but this size is usually sufficient.
Then lightly score the glass side, not the silica side, with the your blade or glass cutter.
It should look like the picture above, with obvious cuts in the glass.
Now carefully pick up the plate and break it by bending it with your hands away from you.
Glass will easily break along a smooth edge if scored.
Be very careful, but you must use enough force to break the glass.
This may take some practice if you have never done it before.
Draw a line in pencil about
0.5 cm from the bottom straight across.If you are doing a simple reaction, as in most undergraduate labs, draw three evenly spaced vertical dashes on the horizontal line.
Label these lines as starting material (SM), co-spot (C), and reaction (R).
This will be explained more in later steps.
Only use pencil to write on the TLC.
If you use pen or marker it will ruin your TLC plate because these media contain dyes which are organic compounds that will develop along with your compounds of interest.
Graphite will not move with the solvent.
Write very gently, otherwise you will scrape off the silica. , A classic solvent system is ethyl acetate and hexane because they mix easily and the polarity of the solvent is easily adjusted.
A good place to start is with 20% ethyl acetate in hexane, which is the same as a 1:4 solution of ethyl acetate to hexane.
You can easily measure out 1 mL of ethyl acetate and 4 mL of hexane in a graduated cylinder, or other liquid-measuring tool, and then combine them to give the desired solution.
In an enclosed jar, place some 1:4 ethyl acetate:hexane so that the bottom of the jar is just barely covered.
Close the lid on the jar, or else the solvent will quickly evaporate and you will have to add more. , Take the starting material for your reaction and dilute it in a small vial with ethyl acetate.
TLC is very sensitive and even a 1% solution of your compound is plenty.
You really only need a very dilute solution for this procedure.
In fact, if your solution is too concentrated, your spots will streak and you will get no useful information from your TLC plate.
If your reaction is also a nice solution you can spot from it directly, but if your reaction mixture is more complex you may have to remove a small amount and dilute it in a suitable solvent (such as ethyl acetate) to make a solution.The best solutions to spot are clear, but they don't have to be.
The color of the solution does not matter.
In the picture above, the solution is clear and colorless because this compound is not colored, but oftentimes the solutions have color to it. , Submerge the tip of a glass pipette into one of the reaction solutions you just made, and the pipette it will suck up a small amount.
This is called capillary action and many people are surprised that this works.
See the above picture for proof that this actually works.
Simply stick the end of the pipette into the liquid, and some of it will come into the pipette.
You do not need very much solution for spotting on the plate, so the tip of a pipette is plenty. , It is very important to spot lightly.
If you press too hard, you will deposit too much material, making big spots which will overlap, causing the TLC to be difficult to read.
This step will take practice, so you may want to try this several times, trying to get as small of spots as possible before continuing onto the next step.
Spot the starting material on the first and second dashes, and the reaction solution on the second and third dashes.
The first dash is your starting material reference (SM), the middle spot is a co-spot (C), and the third spot is the reaction reference (R).
This type of spotting makes the developed plate very easy to read.
The the above picture for visual clarification. , Simply set the plate down and wait a minute or so for the solvent to evaporate off the plate.
You cannot run the plate if there is still solvent.
You will be able to see a wet spot on the plate if there is solvent.
You need not wait long for such volatile solvents as ethyl acetate and hexane.
A minute is more than enough time for these solvents to evaporate. , Using tweezers, gently place the TLC plate into the solvent jar, with the spotted end of the plate at the bottom close to the solvent.
Submerge the bottom edge of the plate into the solvent, allowing the solvent to run up the plate, carrying the compounds along with it.
Cover the jar with the lid.
This will prevent solvent from evaporating and make the development stage much faster.
This is the stage where separation occurs.
So be patient and wait 2-3 minutes for the solvent to reach the top of the plate before you remove it.
Waiting longer usually provides better results, so long as you don't let the solvent flow past the upper edge. , Once the solvent comes close to reaching the upper edge of the plate, use tweezers to carefully remove it from the chamber.
Quickly mark in pencil the highest place the solvent reached.
This is called the solvent front and is important to take note of for later calculations.
Do not touch the silica with your fingers as you could smudge the spots or deposit compounds from your hands onto the plate, making your plate useless. , Again, wait for the solvent to evaporate off the plate.
This should only take a minute or less if you are using a volatile solvent such as ethyl acetate hexane, but it is important to not have solvent on your plate. , Place the plate under an ultraviolet light.
You should notice several spots at different locations, corresponding to different compounds.
The presence or absence of spots corresponds to the progress of a reaction, so information about a reaction can be easily determined in this way.
Use a pencil to outline the spots so you can continue to analyze the plate after you remove it from the UV source.
Look at the above picture and notice the different spots at different heights. , This is simply the ratio of how far the spots have moved to how far the solvent has moved.
Use a ruler to measure the distance to the solvent front and to the middle of the spots.
Divide the distance the spots moved by the distance the solvent moved.
This will give you a number between 0 and
1.
By calculating the Rf of known compounds, you can identify them on a TLC plate.
Different compounds will almost always have different Rf values, making them easy to distinguish.
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Matthew Evans
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