Dear all,

we begin with the analysis of proteins and peptides. Is there someone with experiences in analysis of peptides/proteins? Who can give us
advices to the acquisition of HPLC, detector etc.?

Charly

It really depends upon the size of the proteins you wish to separate. For very large proteins, size exclusion chromatography works well and is very simple, but does not provide high resolution.
Otherwise, a good place to start would be a reversed phase column with a 300 A pore and a gradient between 0.1% v/v Trifluoroacetic acid (TFA) in water and 0.1% v/v TFA in acetonitrile (MeCN). The precise composition of that gradient depends again upon the hydrophobicity of the proteins and the column you use. You'll use less MeCN with low MW peptides or on a C4 or C8 column. You'll use more MeCN with high MW proteins or when using a C18 column. Also, please do note that peptides often do not behave in the same manner as other small organics. Very often an isocratic run is simply not possible with peptides/proteins - even if the matrix is pretty simple.

Detection is done most easily with a UV-Vis (photodiode array preferred).

Vydac specializes in making columns for analysis of peptides on LC, but other suppliers (Waters, Phenomenex, Metachem technologies, Supelco, Keystone Scientific) can also provide some very good columns.

I hope you find this helpful. if I can be of further assistance, please e-mail me at christopher.judd@collabo.com and I'll do what I can.

I have one question... why TFA with Peptides? Why not weaker acids such as formic or acetic acid? Any reason?

Rajesh

works too