Very good tutorial! It's very important to always add the silica gel as a slurry, never dry as some do. Besides that, all surfaces must remain as straight as possible. The sample loading by first adsorbing it onto some silica gel instead of applying it in solution is very interesting, i've never seen that technique! But I guess it's only really needed if the product isn't soluble enough in the eluent to apply with a small volume. Thanks a lot for that! I've got just a few additions: - Better put safety clips on every joint while evaporaring. Losing your product in the rotavap water sucks^^ - Stopper instead of aluminium foil - use those manual pump balloons if you need pressure. Using nitrogen to pressurize the system is always a safety risk. And put a safety clip between the column and reservoir. I've had a liter of solvent splashed into my face because a colleague overpressurized the column. Luckily not really dangerous solvents. With acidic or basic ones I may be blind now. - don't spot on the outer sides of TLC plates. They usually don't flow perfectly straight on the side.
Thank you so much. Is there standard to use silica gel in column, I mean if my sample a little bit like (0.01 gram), how much of silica gel use here? in contrast, if I have a lot of samples such as (1 gram or more), here, how much of silica gel have to use? I mean is there ratio from silica gel to sample to use in column, please tell me
Yes. He showed a little chart. Depending on the different RF factors determined by running a TLC on a silica plate with the same eluent, you'll need about 20 to 100 times more silica gel than product to separate. That chart is a retty good rule of thumb. The RF factor also lets you estimate the total volume of eluent you'll need etc. So it's always better to first find a suitable eluent (and maybe stationary phase. Usually we always use silica gel. But there's also reverse phase silica with C18 chains bonded to it (reversing the whole polarity. So you use more polar solvent for retardation and more organic will elute faster).
If I used wet packing of silica gel to column (slurry), here, can I use any solvent for silica gel until reached slurried, or must use only hexane. thank you
happy new year. If I have solid sample containing components, can I use any solvent to dissolve it? Or I have to use only the same mobile phase as solvent to dissolved sample? Another case, if must use same mobile phase, but my sample can not dissolve, here, please I need solution to this problem. can I use any solvent? thank you alot
No. Because if you use a different solvent than your eluent, this will drastically change and disturb your separation. But he showed a great way to circumvent that problem by suspending it in silica gel, evaporating the solvent off, thus depositing it on the silica gel and loading it onto the column that way! Never seen this before, but it's pretty smart! Btw you'll have the same problem in HPLC. You can't run a reverse phase HPLC with a highly aqueous mobile phase but dissolve your samples in THF. This will tear your peaks apart, since it elutes much faster with that bit of THF (opposite for normal phase). Absolutely destroys the efficiency of the separation.
Theoretically yes, practically, no (as far as I know at least). But for protein separation I'd probably rather use an HPLC and size exclusion columns, if available of course. It of course depends on the properties of the proteins to separate, but usually it's best to separate them by size exclusion HPLC based on their molecular mass.
Exactly, to keep the layer horizontal. Any disturbance in the layer of silica gel will decrease the efficiency of the column, because different heights of silica will cause different separation on each vertical column of your column. Sand is heavy and has much less surface area than silica gel.
Sure, but this will decrease the efficiency (in plate numbers) of your column, because sand gives a better straight layer to hold the silica. But you can keep that at a minimum by only pluggging the narrow end of the fritless column with cotton. If you've got a column with a frit anyways, sand isn't needed. Though it's always good practice to put sand on top of the silica, to protect the silica layer from being disturbed. TL:DR any disturbance of the flatness of your silica layer will decrease the efficiency. Knowing and watching that, you can do a column however you prefer.
Very good tutorial! It's very important to always add the silica gel as a slurry, never dry as some do. Besides that, all surfaces must remain as straight as possible. The sample loading by first adsorbing it onto some silica gel instead of applying it in solution is very interesting, i've never seen that technique! But I guess it's only really needed if the product isn't soluble enough in the eluent to apply with a small volume. Thanks a lot for that!
I've got just a few additions:
- Better put safety clips on every joint while evaporaring. Losing your product in the rotavap water sucks^^
- Stopper instead of aluminium foil
- use those manual pump balloons if you need pressure. Using nitrogen to pressurize the system is always a safety risk. And put a safety clip between the column and reservoir. I've had a liter of solvent splashed into my face because a colleague overpressurized the column. Luckily not really dangerous solvents. With acidic or basic ones I may be blind now.
- don't spot on the outer sides of TLC plates. They usually don't flow perfectly straight on the side.
why is that better to add the silica gel as a slurry and not dry and pack it with solvent after?
Thank you so much. Is there standard to use silica gel in column, I mean if my sample a little bit like (0.01 gram), how much of silica gel use here?
in contrast, if I have a lot of samples such as (1 gram or more), here, how much of silica gel have to use?
I mean is there ratio from silica gel to sample to use in column, please tell me
Yes. He showed a little chart. Depending on the different RF factors determined by running a TLC on a silica plate with the same eluent, you'll need about 20 to 100 times more silica gel than product to separate. That chart is a retty good rule of thumb. The RF factor also lets you estimate the total volume of eluent you'll need etc. So it's always better to first find a suitable eluent (and maybe stationary phase. Usually we always use silica gel. But there's also reverse phase silica with C18 chains bonded to it (reversing the whole polarity. So you use more polar solvent for retardation and more organic will elute faster).
If I used wet packing of silica gel to column (slurry),
here, can I use any solvent for silica gel until reached slurried, or must use only hexane. thank you
Yes, you can use any solvent. It must be your eluent though.
thank you@@GodlikeIridium
Stupid question: what is the reason of putting a layer of sand on top of the frit at the bottom of the column?
It's not a must, but I prefer having it to avoid clogging the frit. It also makes it easy to empty the column at the end.
happy new year.
If I have solid sample containing components,
can I use any solvent to dissolve it?
Or I have to use only the same mobile phase as solvent to dissolved sample?
Another case, if must use same mobile phase, but my sample can not dissolve, here, please I need solution to this problem. can I use any solvent? thank you alot
No. Because if you use a different solvent than your eluent, this will drastically change and disturb your separation. But he showed a great way to circumvent that problem by suspending it in silica gel, evaporating the solvent off, thus depositing it on the silica gel and loading it onto the column that way! Never seen this before, but it's pretty smart!
Btw you'll have the same problem in HPLC. You can't run a reverse phase HPLC with a highly aqueous mobile phase but dissolve your samples in THF. This will tear your peaks apart, since it elutes much faster with that bit of THF (opposite for normal phase). Absolutely destroys the efficiency of the separation.
Can this technique be used to separate proteins in blood?
Theoretically yes, practically, no (as far as I know at least). But for protein separation I'd probably rather use an HPLC and size exclusion columns, if available of course. It of course depends on the properties of the proteins to separate, but usually it's best to separate them by size exclusion HPLC based on their molecular mass.
Why are you putting the last layer of sand before putting the crude mixture ?
thank you
I believe that's just to kinda hold down and stabilize the top layer of silica. You don't want it moving at all when you pour the eluant onto it.
Exactly, to keep the layer horizontal. Any disturbance in the layer of silica gel will decrease the efficiency of the column, because different heights of silica will cause different separation on each vertical column of your column. Sand is heavy and has much less surface area than silica gel.
thank you so much!!!
Can i use cotton instead of sand
Sure, but this will decrease the efficiency (in plate numbers) of your column, because sand gives a better straight layer to hold the silica. But you can keep that at a minimum by only pluggging the narrow end of the fritless column with cotton. If you've got a column with a frit anyways, sand isn't needed. Though it's always good practice to put sand on top of the silica, to protect the silica layer from being disturbed.
TL:DR any disturbance of the flatness of your silica layer will decrease the efficiency. Knowing and watching that, you can do a column however you prefer.