February 1, 2023

Thin layer chromatography (TLC) analysis was performed to determine the identity of an unknown compound. The known possible compounds were acetaminophen, acetylsalicylic acid, caffeine, and salicylamide.

In this lab we’re doing tlc analysis to figure out the identity of an unknown compound the four possible compounds this week are caffeine acetaminophen salicylamide and acetyl salicylic acid tlc stands for thin layer chromatography it’s a separation technique that uses the difference in polarity of our samples to separate them so they have two parts we have a mobile

Phase and a stationary phase our stationary phase is the silicon dioxide layer on the tlc plate itself and our mobile phase is the liquid we’re putting it in this week it’s going to be 95 to 5 ethyl acetate to acetic acid so we have a polar stationary phase and a non-polar mobile phase this is called normal phase chroma tlc reverse phase would be if you switch

Them if you had a non-polar stationary phase and a polar mobile phase so in this case the liquid is going to travel up the silicon dioxide layer and our compounds are going to separate based on their absorption to the stationary phase so the more time they spend absorbed the stationary phase the less further will travel up the tlc plate at the end we’re going

To calculate an rf value which stands for retention factor this is the distance the product moved divided by the distance the solvent moved which will will compare to the known products and figure out what our sample is first we’ll need about five to ten mils of our mobile phase in this case it’s 95 percent ethyl acetate five percent acetic acid and a beaker the

Size of the beaker doesn’t really matter as long as you can fit the tlc put in there i’m using a 250. and the amount you’ll need will vary based on the beaker but you want about less than a centimeter of solvent in there and then we will cover it with some aluminum foil that’s a good amount we’ll go get that with the foil all right if you want you can put a piece

Of filter paper in there to help saturate a little bit most time it’s not needed though the aluminum foil should work just fine all right in this lab there’s several pre-made unknowns i at the moment don’t know what any of these are so i’m just going to go ahead and select one and that’s what we’re going to use today so this is tlc unknown 16. so now our goal is

To figure out which one that is all right so here’s our tlc plate this white layer on top or you’ll notice there’s two layers to it this white layer on top is our silicon dioxide layer and this reflective layer is just some aluminum so when we’re spotting we have a roller next to it so that when we’re spotting our solution we make sure we’re high enough if it’s

Too low and it’s in the liquid instead of the liquid coming up the tlc plate to where it is it will disperse the liquid you won’t get you’ll smear and you won’t get a good tlc plate so it’s really important to do it above where your solvent is in your container in our container right now measured our solvents about half a centimeter or so so we’re gonna put this

About a little bit above the one centimeter mark when you’re marking on these you can use a pencil do not use a pin a pin has compounds in it that will travel up the tlc plate and mess up your tlc so we’re going to go ahead and mark this just on the side all right one centimeter all right you want to mark it lightly so that it doesn’t scratch up the surface too

Much and if you do scratch a little bit just make sure your sample is not on the scratch part you can also put little reference dots of where you want your samples to be what i’m going to do with my samples is i’m going to put my unknown on the far left and then i’m going to go alphabetical order so it’s going to go acetaminophen acetyl salicylic acid caffeine and

Salicylamide just an easy way to remember whatever way you do it is fine just make sure you keep track of which unknown is which i’m not going to put reference dots on there but you can just put a little tiny reference dot of where you want each of your samples to be our next thing is to dissolve all our samples in some one-to-one dichloromethane and ethanol and

Then we will spot them on the plate with some capillaries i got some test tubes here i’m just going to put a few milligrams i’m not going to measure it into these and a three to five drops of our one to one dichlomethane ethanol solution and then we will spot them on the plate all right so i have each of my samples and a couple drops of the one to one dichlomethane

Ethanol and next we’re going to use some kepler tubes in order to spot them carefully on the plate you don’t want a giant spot you just want enough but it’s on there so one dab or so on each is probably fine and afterwards we can check with the uv light to make sure that they’re all on there it’s also very important to make sure that they’re all about the same

Level so that when you’re doing your measurements later they will be accurate all right so first our unknown sample now for the acetaminophen now for the acetyl salicylic acid now for the caffeine and finally the salicylamide okay so they’re all on there we will plug up the uv light use some shortwave uv light to check to make sure they’re all on there

All right so now you can see on the camera we got some shortwave 254. nanometer uv light and all of our samples are on there they’re well spaced apart the one two on the right here a little close but we’ll see how it develops our next step is to put it carefully into the solution all right so now that we have our sample spot on our tlc plate we’re going to take the

Lid off here or a beaker now in my opinion this is probably my most nerve riding part because i can get some shaky hands but you want to carefully put it in i recommend using some tweezers or something in such a way that again none of your samples go below the solvent line and it enters all evenly at the same time you enter it this way they’ll skew and travel

Not straight up the plate so you just want to carefully put it in if you do mess up you just remake a new plate it’s okay but you just try and carefully put it in all at the same time it’s pretty good you let it stand up against the side and now we put our lid back on carefully carefully not to disturb the tlc plate so i’m not going to put it on too tightly

And now we will wait until it’s about three quarters of the way up all right so we’re about three quarters of the way up so i’m gonna again carefully just remove it from this from the beaker now you can see about three quarters of the way up and then first thing i’m going to do is mark where the solvent is right at this moment all right so the next step we’ve

Marked where our solid front traveled to you then want to use the uv light to mark the shape of your where your spots are we got a pretty good development here none of our spots smeared together if they do you may need to redo it but you’ll want to take note of the color shape and how far it traveled up the tlc plates so we’ll go ahead and get marks around where

All of our salt where all of our samples are all right so we have marked out where our samples are and we have three up near the top all look like they’re in a row together we will calculate the r value our values at the end but another thing to pay attention to when you’re looking at your sample under uv is color so sometimes you can have samples that are really

Close in polarity and if we let it develop a really long time on a really big plate they may have separated but with the limitation of the plate we have they’re all still quite close together in this case the next thing you want to look at is the color so we have two three p three dots up there v2 on the outside are both purple and the one in the middle is like a

Reddish brown the fact that our so remember our sample is on the left it is purple and our salicyl mind on the right is purple as well so this identifies our unknown compound as salicylamide when you are measuring your rf values you measure from the start down here at that line where you dotted them all and you measure to the middle of your spot all right so now

We’re going to make our measurements again start from where your solvent line started so our solvent traveled four point two centimeters and we’ll use that to compare to all of our samples are unknown traveled four i’ll say exactly four centimeters are cinnaminophen traveled 3.3 centimeters our caffeine traveled 4.1 centimeters sorry our acetaminophen

Travel or caffeine all right so now we will make our measurements of our for our rf values i’m gonna move around here so i can look at it well all right so our solvent traveled 4.2 centimeters are salicylic acid actually so now we’re going to get our rf values i’m going to go ahead and extend this line of where the solvent started cross just using again

A pencil try not to scratch but at this point it’s okay if it does a little bit more all right so now we will get our rf values r now we will get our distances for rf values so our solvent traveled 4.2 centimeters our salicylic acid traveled 3.9 centimeters our caffeine traveled 1.6 centimeters are acetyl salicylic acid traveled three point eight centimeters

And our acetaminophen traveled 3.4 centimeters and our sample traveled 3.9 centimeters next clip i will show you walking through the math how to get your rf values and we’ll compare our unknown to the four compounds and further confirm it is salicylamine our distance is up the top left just again to remind you we have the sullivan at 4.2 centimeters unknown at

3.9 and then our known samples you’ll see them independent 3.4 acetyl salicylic acid at 3.8 caffeine at 1.6 and salicylamine of 3.9 so the formula for retention factor or rf value is our distance of smaller moved divided by the distance the solvent moved all right so just take our distance our spot moved and divided by 4.2 centimeters each time so for our unknown

We get 0.93 for acetaminophen we get 0.81 for acetyl salicylic acid we get 0.9 for our caffeine we get 0.38 and for our salicylamide we get 0.93 so this confirms our unknown and salicylamide

Transcribed from video
Thin Layer Chromatography (#5) By Cameron Flester – EKU Chemistry Grad