I am trying to separate plant metabolites by atmospheric pressure column chromatography with silica gel (normal phase), packed column carefully and equilibrated. The column was non-fritted and used a cotton ball to plug the bottom of the column. Initially the flow rate 0.5ml/min and I did not change the flow rate but suddenly the flow stopped completely and observed few bubbles inside column. Can anyone correct the fault or it is common?
Hi Ali,
I'm not sure if the link solves your question, but worth to read it in your case.
Good luck,
Marcus
http://www.chemistryviews.org/details/education/2040151/Tips_and_Tricks_for_the_Lab_Column_Packing.html
Marcus Binner
Thanks for your information. It will help me a lot. But they did not provide clear information about bubble formation and cracking of the column. It would be appreciative if you provide some information regarding this.
Thanks again.
Ali Reza
What solvents were you using? Were you running isocratic or stepped gradient?
I've observed this with columns packed in hexanes with gradients. Upon the step to ethyl acetate, the ethyl acetate adsorbs onto the column and displaces bubbles (I don't know it it is nitrogen or oxygen being displaced). The column also gets a little warm from the heat of adsorption of the ethyl acetate. A hexane/dichloromethane gradient behaves the same way. This happens also with petroleum ether, heptanes, or cyclohexane.
The way to avoid the problem is to pack the column in 1-2% "B" solvent. The B solvent is the strong solvent which causes the compound(s) to elute (ethyl acetate or dichloromethane in my examples above).
Mr. Jack Siver
I have been using Di-choloromethane as solvent and running stepped "gradient elution".
By the way thanks for your valuable suggestion.
Ali Reza
One other thing- if the column was "dry-packed", and the dichloromethane flowed through the packed column, the same issue could occur. You may try "wet-packing" the column as a slurry, if you aren't already doing so.
Mr. Jack Silver
Thanks again.
For your kind information i have tried "wet packing" column.
Oh yes, packing with a slurry is much better in terms of uniform column packing. Pre-mix the dry stationary phase carefully with the solvent of choice, then add to the column. Wait a bit to let the stationary phase settle, then remove excess solvent with slight pressure from the top, leave 5-10 mm solvent above the stationary phase. Then, I always used a cork ring with which I gently hit the column from the outside which ensures that the stationary phase settles further and compresses better. In between, remove solvent with pressure and so on.
Now repeat the above mentioned steps until the column is tightly packed with the amount you want.
Jack's recommendation is gold, if you use a small amount Ethylacetate or related solvent you use for gradient, this prevents heating upon adsorption of the more hydrophilic solvent during gradient (= stronger interaction with silica = heat release (adsorption enthalpy) = evaporation of hexane or dichloromethane = bubbles)
Marcus
Marcus
Thanks for your expertise suggestions. Your information helps me a lot and successfully doing my column.
Hi Ali,
great to hear that, wish you best success for your research!
Marcus
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