peak separation

[vanja13]

Here is chromatogram
http://i19.tinypic.com/30j1rg7.gif

First component is desmedipham, then phenmedipham and ethofumesat.
Eluent is 50% MeOH/50% water, flow is 1,3 ml/min.
Column is Supelcosil lc 18 5 um,150 mm x 4,9 mm.
Hplc, UV detector 225 nm.
How can I get better separation of 2nd and 3rd peak?

thank you

[Rob Burgess]

If you still want to run isocratically, try dropping your % organic down to gain more retention and hopefully better separation. You could also alternatively try a different mobile phase composition i.e. using acetonitrile instead of methanol. Try altering the pH of your mobile phase as well if your compounds are acidic or basic. Finally you could also try a gradient separation, assuming your HPLC system is capable to do this.

[danko]

In addition to Bob’s excellent suggestions, I’d say: Flow rate of 1.3 mL/min is a little bit high for 5 μm particles (4.9 mm column diameter? Are you sure it is not 4.6?). Not all too high, but I’d guess that 1.0 mL/min might result in better resolution.

Best Regards

[vanja13]

(4.9 mm column diameter? Are you sure it is not 4.6?).

You are wright!
I wrote it wrong, column diameter is 4.6 mm.

Thanks for your help!