HELP !!! packing C18 column

[felixyong22]

hi, good day to everyone here. i m newbie here and currently i bought a LiChroprep RP-18 (40-63µm) powder, and started to pack today. i got many questions to ask regarding this RP-18 column because i really lack of knowledge on this fractionation thing and i hope you all can help me out.
the questions are:

1. i used water:acetonitrile (7:3) as solvent to mix with the RP-18 powder, but it seems the powder floating on the solvent and formed lot of bubble. is it my solvent system got problem? or i use the wrong solvent?can anyone help me on this?

2. the flow rate is quite fast from the beginning when i pour the slurry inside the glass column, and the flow rate become slower and slower. it takes 5-6 seconds to drop out 1 drop. but the length of the powder inside the column is just 15cm height. it is still need to pack more right if not mistaken? i took 2 days to pack it, but still not yet finish to pack til 1/2. can anyone help me to solve this problem?

3. basically is there any standard ratio on how tall we should pack for the column or just depend on our preference??

hope to get all the expertise's answer here, bcoz i really confuse and frustrated on what i m doing now..thank u so much... thanks a lot...

[Uwe Neue]

1. You need to pack the column in a solvent that wets the C18. Your choice of solvent was wrong: no water!!!! You can make a slurry with neat acetonitrile, and it will work better.

2. The resistance to flow will increase as the bed builds up. To avoid the initial high flow, you can put a valve or a restrictor tubing at the outlet of the column. Not sure why it is so slow at the end. Could be fines in the powder, or a clogged column outlet frit. See if the frit is compatible with the particle size.

[Chromatographer2010]

The lack of flow could also be related to the wetability of the powder. If the liquid wets out the C18 better then the flow will probably be improved.

Also with your particle size you could probably get away with dry packing the column. Basically you just tap and rotate the column as you put small amounts of the dry packing into the column. Repeat until you get your desired bed depth.

I googled dry chromatography packing and got about 8 references that describe the process including "Introduction to Modern Liquid Chromatography" Snyder, Kirkland and Dolan.

[felixyong22]

i need to have use water in my packing bcoz my compound only can totaly dissolve in water but not acetonitrile. and somemore, i consulted with technician and also my supervisor, they said as long as not 100% of water to pack RP-18, then will be fine. is it need a vacuum pump for example peristaltic pump to pump it so that the flow rate can be increased?

[Chromatographer2010]

You will be using water when you do your analysis with your compound but that doesn't mean you need to use it when you're packing. Just switch over to you mobile phase (with water) when you start doing your analysis.

The fact that your powder is floating on the solvent and there is air trapped around the packing material when you mix it with the 70/30 H2O/ACN indicates that your packing is not getting wetted out and that it is not a good candidate for your packing solvent.

Yes, applying pressure to the inlet or vacuum to the outlet will increase the flow.

[Bintang]

For questions regarding Merck chromatography products please contact us at
chromatography@merck.de
we will help you with your packing problems.