Chromatography Forum

I am working of detecting protein ligand complex. Can I inject the sample directly in my C18, 4.6x150mm,3.5micron column, assuming that ligand will separate from protein as the organic solvent will kick in and i can detect it.Will the protein destroy my column....can somebody help me i am very new to this protein thing.

Proteins are routinely analyzed with RPLC so its unlikely your protein will damage your particular C18 column. Be sure to elute the protein from your column to avoid plugging. I have run myglobin using an Onyx monolithic C18 column (50x2.0mm) and a gradient from 5-95% ACN with 0.1% formic acid without problem. The heme and the apomyglobin were well resolved.

A wide pore material (300A) may be needed depending on the size of the protein your analyzing. If the protein is too large for standard HPLC media (~100A) it will simply elute very early or unretained.

Hope this helps

Carl

It also depends on the nature of the protein. If the protein is very hydrophobic (has many hydrophobic residues) a C18 column may be too hydrophobic and you may have difficulty getting it off the column. From what I understand, C8 columns are more frequently used with whole proteins. Also ion exchange, and size exclusion are also frequently used with proteins though more so for preparative applications.

I've worked with membrane binding proteins that were so hydrophobic that putting them in just about any column was like the protein bermuda triangle (It disappears and is never seen again).

Dear,

Try ZICHILIC COLUMN FROM MERCK WHICH IS VERY SUITABLE FOR PROTEIN SEPARATION THIS COLUMN IS FROM MERCK