Відео

why is that better to add the silica gel as a slurry and not dry and pack it with solvent after?

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.

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.

Yes, you can use any solvent. It must be your eluent though.

thank you@@GodlikeIridium

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.

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).

why are you increasing the pressure if you want to evaporate a solvent?

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.

Price varies a lot. It can go up to 20k GBP.

Nah only like 3k now

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.

Thats what im thinking right now

Yes it depends on what your product is but generally you'd use a warm solvent e.g. Ethyl acetate, which is then cooled to give you crystals that crash out upon cooling

It really depends on what you need to do next. If your product is a solid you can scrape it out with a spatula, if is a liquid you pipette it out etc.

Haha! :P