Sciencemadness Discussion Board - Preparing a chromatography column

Preparing a chromatography column

Boffis - 17-6-2020 at 01:44

I have never used column chromatography before but following a recent experiment in which I tried the benzal acetone reaction with salicyladehyde and acetone I have run out of alternatives. The reaction gave a mixture of a pale green and a reddish brown crystalline products. I have managed to manually pick out some of the pale green material and recrystallise it separately to obtain fairly pure pale green crystals but now the residue always gives intergrown crystals that can't be hand picked. I have tried numerous solvents but their solubilitys are very similar so I thought I would try column chromatography on some of the mixture. I have several columns with glassy frits in the bottom and taps below and I have a large tub of chromatography grade silica gel.

The question is how do you actually construct the column? How do you stop the silica from washing through the frit (its a very coarse frit) and how do you prevent bubbles forming in it? Has any one got any first hand experience and can point me in the right direction.

DavidJR - 17-6-2020 at 02:33

How fine is your silica gel? you shouldn't need to worry about it passing through the frit if you're using the normal kind used for column chromatography (70-230 mesh).

As for packing the column:

Close the stopcock and attach a funnel to the top of the column
OPTIONAL: Pre-fill the column with some solvent (I prefer not to do this but some people like to do it this way).
Prepare a slurry with a suitable quantity of silica gel and small amount of solvent (starting mobile phase)
Stir to suspend the silica and quickly dump the slurry all at once into the column
Rinse the walls of the column with solvent
Tap the column if necessary to dislodge any bubbles and settle the silica
Ensure the silica top is flat and level and top it with some acid-washed sand (optional) to avoid accidentally disturbing the silica gel later
Open the stopcock to run the solvent down to just at the top of the silica gel

Then you're ready to load the column and elute. Make sure not to let air be drawn into the silica gel bed because when you add more solvent it doesn't effectively get rid of the trapped air and your separation might go to shit.

Ubya - 17-6-2020 at 02:47

there are a few ways to pack a column, i would suggest the wet method.

mix your silica with enough solvent (keep track of hoe much you are adding)to form a thin slurry (if you stop mixing it settles so be quick when pouring), close the stopcock and pour the slurry down the column (be quick so the silica settles uniformly), wait for it to settle on its own and then use a soft rubber tube to gently hit the column to pack the silica a bit more.
drain the solvent until the meniscus is right at the end of the silica column (never go below), keep note of the volume of solvent you get, the volume of solvent you added to form the slurry minus the solvent you got now gives you the amount of solvent held in the column (when you later elute your compound you can ignore this volume of solvent eluting through the column at the beginning as it won't have any of your compounds).
add your compound mixture dissolved in a minimal amount of the same solvent you are using as eluent, open the stopcock and again lower the meniscus at the level of the silica (you can do this multiple times), now add the solvent slowly from the wall of the column to not disturb the top layer (you can also add a layer of sand on top of the silica to protect it from the solvent falling in the column). you can start the elution process

there are many youtube videos that show you the packing process

Boffis - 17-6-2020 at 02:49

@DavidJR, many thanks for this info. Yes my silica is Silica gel 60 and 70-230 mesh. I haven't decided on solvents yet I am running some tlc first to see what sort of solvent will be best.

My column has an ID of about 16mm at a guess any idea what sort of loading I can apply in terms of how much of my mixture I can treat in one pass?

Ubya - 17-6-2020 at 03:24

DavidJR - 17-6-2020 at 03:30

It does depend on how good a separation you get though. If well resolved you might be able to exceed the loading on Ubya's graphic by a considerable margin.

Housane - 18-6-2020 at 02:04

Could a burette be used as column? Or should i buy one?

brubei - 18-6-2020 at 02:32

You can do it in a syringe or pipette too.

the only limitation is the ratio of silicagel:product quantites. In my memeories you need 20 to 50 times more silicagel than product + impurities

Ubya - 18-6-2020 at 03:48

Quote: Originally posted by Housane

Could a burette be used as column? Or should i buy one?

a burette is pretty thin and long, a normal 10ml pipette should have a similar diameter, but it's way shorter.

Eddie Current - 18-6-2020 at 04:53

Quote: Originally posted by Housane

Could a burette be used as column? Or should i buy one?

No. You will only end up blocking the burette making it useless.

Burettes are typically utilised for titrations.

You need to read/watch more video's of chromatography to gain a better understanding.

Housane - 18-6-2020 at 05:11

Yeah I know what a burett is used for his seeing if I could use it for another thing @Eddie Current

SWIM - 18-6-2020 at 11:23

Quote: Originally posted by Eddie Current

Quote: Originally posted by Housane

Could a burette be used as column? Or should i buy one?

Why would it block up if you use glass wool and sand to keep the packing out of the valve?

I believe this was done on old columns that lacked a frit.

JJay - 18-6-2020 at 17:20

I have certainly read of burettes being used as columns. (See, for example, https://pubs.acs.org/doi/full/10.1021%2Facs.jchemed.8b00665)

Mateo_swe - 19-6-2020 at 01:54

I think it more depends on the size of the silica used and not the burette/column.
If you have a very fine silicagel, like TLC silicagel 15µm, it might not work properly and get blocked.