Protein injection in C18 column

[mk12]

Can I inject protein (size29000D) in my RPC18, 4.6x150,3.5 micron column. Is not going to destroy my column.

[kerri]

What brand of column is it? I am no expert with RP protein chromatography, but I do know that the pore size can make a difference.

Kerri

[tom jupille]

Depends on how much you are injecting and, as kerri suggested, on the pore size of the column. Particle size is a non-issue.

If the protein is one of your analytes, then C18 columns generally are not the first choice. Many C18 columns are sufficiently hydrophobic that proteins are irreversibly retained. By the time you get the organic solvent concentration in the mobile phase high enough to displace the protein the protein becomes insoluble. A shorter-chain ligand on a wide pore (around 300 A) is the usual choice.

If the protein is a contaminant, then it will probably accumulate. If the level isn't too high, you can use a guard cartridge or periodically flush the column with a chaotrope.

[mk12]

I am analyzing protein ligand complex. Purpose is to identifiy the small molecules which binds to protein. I can not do protein ppt step to remove protein cause it will dilute my sample. So i am injecting the whole complex, assuming that organic solvent will break the affinity and small molecule will pass through the column and protein will remain at column head. My column is water xrbride, C18,4.6x150mm, 3.5 micron 100A pore size. Few injection should be ok but i am worried later it will clog my column. will the protein size 29000D will enter the this size column or will stay at column head.

[tom jupille]

You protein will probably precipitate/bind at the head of the column. If you have the small molecule separation worked out already, and if you will not be injecting thousands of samples, a guard cartridge may be the most cost-effective solution. Buy a half-dozen and change the cartridge when the pressure gets too high or the separation deteriorates.