sample-concentrations for peptides or small molecules (hplc)

[Dualis]

hi everyone,

i'm starting with rp-hplc. i have a rp18 grom-sil 150mm x 4 mm (5µm) column and will run a gradient from water to acetonitril. detection will be done by uv-vis 215 nm or 220 nm.

i will do enzymatic kinetic research, so i will have different concentrations of my substrates and product. the catalized reaction is like A (a tripeptide) + B (a molecule of 335 g/mol) -> C.

and now i need to know the concentration-range (detection limits) for the hplc before starting. does anyone have any suggestions or experiences?

thank you!

[Kostas Petritis]

Assuming that you have enough resolution between your compounds, I would probably start with the highest possible concentration of my substrate (i.e. at the point just before over-loading the column). It all depends also of the response of your products at the wavelength you will be monitoring. The idea is to be able to have the highest dynamic range possible (i.e. to start detecting your products even at the lowest concentrations at 0.1 to 1%).

[skunked_once]

You may want to include some organic modifier to your mobile phases if you are separating peptides. Trifluoroacetic acid at 0.1% is often used.

[Dualis]

You may want to include some organic modifier to your mobile phases if you are separating peptides. Trifluoroacetic acid at 0.1% is often used.

thx, i already considered that, just didn't write it down here