Peptides Separation - Early Elution

[MissMaia]

Hi, guys!

I'm a new member and also a new HPLC user.
I'm working on an academic project that aims to separate 7 small peptides.
One of these small peptides is being eluted very early (in the void volume). I have already made some changes in the gradient curve and some method parameters trying to delay the first peak elution, but nothing has worked.
Actually, it's worked once (5-65% Mobile Phase B), but it is not reproducible.

RP-HPLC
Shimadzu System
Column Phenomenex C8 250 x 4.6 - 5 micron 100 Angstrom
Method parameters:

Injection Volume: 30 μl
Mobile Phase A: H2O + 0.1% TFA
Mobile Phase B: ACN + 0.1% TFA
Gradient : 10 - 65% MP B


Could you please provide me with advice on how to change it?

Thanks in advance!

[ChristofferBR]

Are you running a linear gradient between A and B?

If so, maybe it might be worth trying a gradient closer to logarithmic - or two consecutive linear gradients, the first one much flatter than the other.


--Chris

[tom jupille]

One of the downsides of gradients is that the column must be re-equilibrated with the initial mobile phase before a second injection can be made. If your 5% initial gradient worked once but was not reproducible, it's possible that the column was not fully equilibrated after the first run. A good "rule of thumb" for equilibration is "10 column volumes plus the dwell volume". A 250x4.6mm column has an internal volume of about 2.5 mL. You will have to measure the dwell volume (gadient delay volume) of your system. More information on dwell volume issues is available here: http://lcresources.com/tsbible/31062013.PDF