Chromatography Forum

Hi, I'm trying to separate a mixture of protein and vitamin D. The protein is amphiphilic and charged, the vitamin D is highly hydrophobic but dispersed in small particles.

I tried using an anion exchange column to "stick" the protein and allow the vitamin D to run through, but in blank experiments with just vitamin D I see no UV absorbance coming out of the column.

I suspect it's due to the PE and PS parts within the column. Are there any columns - perhaps made of glass - that would be compatible with my experiment?

Uhh, some details on the column and instrument you are using would be helpful.

That said, my first thought would be to simply precipitate the protein by adding ACN, centrifuge, and then run the vitamin D on any of the myriad reversed-phase columns that have been used.

I'll be more specific:

I've been working with a GE HiTrap SP HP ion exchange column (1 ml column volume). My sample is a protein in aqueous solution, to which I add fat-soluble vitamin D, in 100% EtOH; the final sample is 2% EtOH. Some of the vitamin D binds to the protein, and some doesn't. Because vitamin D is highly hydrophobic, the free population tends to aggregate into particles which are often larger than the hydrodynamic radius of the protein itself. That's why I can't simply filter the two to separate them.

Because the protein carries a significant surface charge, but the vitamin D particles are either uncharged, or significantly less charged, I thought I'd use an ion exchange column to trap the protein (and the vitamin bound to it), while allowing the uncharged vitamin to pass through.

However, when I tried running a sample including only vitamin D, I did not see the characteristic UV peaks that I saw when I ran the same sample through the system with no column attached.

Since the column contains PS and PE parts, I am guessing that my vitamin is getting stuck there. I'm wondering if I could find a system, maybe constructed of metal or glass, that would not cause this problem. I realize it's a little unusual to be looking for an ion-exchange column compatible with hydrophobic compounds, but that's what I'm looking for.

Because I don't want to disrupt the system, that rules out ACN.

sometimes it is not possible to run everything together
you could go using SEC
but then you will not see D2 and D3 they will come out as a mix
D is a small molecule
it will be hard to see d2 and d3 while using a big pore size column due to the protein
depending on the protein size maybe a 160 or 200A column will work but a 300A will not
we tried it in the past
we decided to separate the chromatography
one method for the proteins, one method for D2 D3

for vitamin D, any ordinary C18 or C8 will do