I pack it just like silica gel. I slurry-packed it in water and attached the column to my flash chromatography system (CombiFlash Rf-200), I ran a gradient starting with water to 100% methanol, another gradient from methanol to methanol to 100% acetone, then acetone to 100% water containing 5% ammonium hydroxide (3 gradients total). The 3 gradients eluted different compounds.

if you use a glass column, this can be done with step gradients as well with the same solvent systems. Running the multiple gradients didn't cause any issues with swelling or back pressure.

Good morning Rania. Polyamide columns are useful to separate groups of compounds in a mixture, usually dissolved in an aquous solvent. First step, you should swell your polyamide in the buffer you choose for separation. Then, you must pour the swelled polyamide onto the column, in such a way that air bubbles don't became trapped insede the filling. Then you must let the filled column to be equilibrated with the initial buffer, by adding small quantities of buffer, that go through the polyamide filling, and go to waste at the end of the column. After the equilibration step, you put your sample in the top of the column (previously equilibrated in the same buffer), and you can detach the first fraction of compouds with a solvent with other polarity (e.g. acidified methanol, or only methanol). Then, you can colect these fraction at the bottom of the column.

You must read the bibliography for details of solvents/buffers to be used.

I pack it just like silica gel. I slurry-packed it in water and attached the column to my flash chromatography system (CombiFlash Rf-200), I ran a gradient starting with water to 100% methanol, another gradient from methanol to methanol to 100% acetone, then acetone to 100% water containing 5% ammonium hydroxide (3 gradients total). The 3 gradients eluted different compounds.

if you use a glass column, this can be done with step gradients as well with the same solvent systems. Running the multiple gradients didn't cause any issues with swelling or back pressure.

According Ndjoko (2010), they used polyamide columns to dereplicate tannins. They dissolved the extract in EtOH 96% and applied in 0.5 g of polyamide for each 65 mg of dry extract. The washes consisted in EtOH 96% (twice). http://onlinelibrary.wiley.com/doi/10.1002/pca.1262/abstract

Dr Jack silver, I'd like to know if you use a constant volume of each mobile phase?? or just use TLC analysis to change the mobile phase ??

If constant volume how could you determine the volume to be used??

In my case, I used a constant volume for each gradient, around 20 column volumes. If running a glass column (step gradient), I'd use 10 steps with 2 column volumes per step.

Okay.. Does the sample have to be completely dissolved in the first mobile phase (water) ??

Good morning Rania, If your sample is not completely dissolved in the first mobile phase (in your case, water), is not very bad, if you can be sure that the compounds you want to separate in the column, are dissolved. For instance, if you have a mixture of polysaccharides with tanins to be separated, its normal that (using water as solvent), the polysaccharides will be completely dissolved, and the tanins only partially dissolved.

Then, when you put the partially dissolved sample in the top of the column, the non-dissolved tanins stay in the top of the column, and only the dissolved compounds go through the column.

It's helpful if everything gets dissolved. If running a gradient, the undissolved compounds may take some time to actually dissolve in the mobile phase and create poor chromatography.