Gradient HPLC, need some suggestion?

[sophieUMB]

I have two compound, one is eluted much faster than the other, so i choose gradient setting.
Here are the descriptions
1. Mobile phase, A 20 mM Ammonium acetate; B Methanol
2. C18 column, 220*4.6mm, 5micron
3. flow rate 1.0ml/mlm
4. One compound, PKa=6.5, elute at around at 5min when mobile phase A:B=60:40, it is hydrophilic
5. The other one, PKa=7.75, elute at 12min when mobile phase A:B=40:60, hydrophobic compound.

Here is my gradient setting based on the above instrument,
Flow MIn A B
1ml/min 1 60 40
1ml/min 8 60 40
1ml/min 10 40 60
1ml/min 15 40 60
1ml/min 17 60 40
1ml/min 20 60 40.

May I run HPLC like the setting above to get these two analytes run in the same methods.

Thanks,
Sophie

[Consumer Products Guy]

May I run HPLC like the setting above to get these two analytes run in the same methods.
Thanks,
Sophie

I don't see any reason why not. I do see where your method has only three minutes, at 1ml/min as time at the initial 60/40 mix - so 3.0 ml worth. So I'd add a post-run time to allow for additional column re-equilibration, maybe 5 minutes. Such post-time will make your retention times more consistent, if your 3 minutes wasn't enough to re-equilibrate.

[LiVD]

Flow MIn A B
1ml/min 1 60 40
1ml/min 8 60 40
1ml/min 10 40 60
1ml/min 15 40 60
1ml/min 17 60 40
1ml/min 20 60 40.

May I run HPLC like the setting above to get these two analytes run in the same methods.

Given the fact that you've only got two analytes to separate, you'll probably be able to shorten your run times. I would try something along the lines of:

Flow MIn A B
1ml/min 1 60 40
1ml/min 2 60 40
1ml/min 10 20 80
1ml/min 11 20 80
1ml/min 12 60 40
1ml/min 17 60 40.

After which you could further shorten it by reducing the gradient time and increasing flow rate (within the limitations of your pump of course).