Chromatography Forum

Hi

I am trying to do a scouting gradient to determine if I can use isocratic conditions for my HPLC run, unfortunately I have peaks coming off after my gradient has finished and i cant find anything online to help. conditions are below.

column: Hichrom Halo C8 150mm, 5μm
run time: 20 mins
solvents: A- Acetonitrile + 0.1% Formic acid. B-Water + 0.1% Formic acid
Gradient: 5 - 95% Acetonitile

Any suggestions on how i can get all my peaks to come off within my gradient would be helpful.

Thanks

You can either increase the temperature, the flow or use a stronger eluent. 50:50 ACN:IPA as organic works well. Pressure will be higher though.

Hi

what means "after the gradient"?
after the programmed gradient time but still within the gradient delay time?
Then the peaks are eluted still within the gradient.

If they're in the plateau at 95% after gradient finished, then your molecules are too lipophilic for this gradient and you may need more elution strength. (or less lipophilic stationary phase).

First thing to try may be to let the gradient go up to 99% or 100% B.

If that is not working, maybe you can add something with higher elution strength on reversed phase. Maybe add some % of isopropanol to the acetonitril (never tried but; watch the pressure as IPA is more viscous. To some extend, even ethyl acetate may be possible, as long as it is miscible with the aquoues solvent).

On the other hand, if all your peaks elute that late in the gradient, you may start at higher %B (e.g. 50->95 or 75->95) which makes the gradient more shallow. This may also help.

If the peaks are already widely distributed over your gradient, then you probably already have your answer and isocratic is not the way to go (which then leaves you still with the same issue on the gradient separation).

Hi

what means "after the gradient"?
after the programmed gradient time but still within the gradient delay time?
Then the peaks are eluted still within the gradient.

If they're in the plateau at 95% after gradient finished, then your molecules are too lipophilic for this gradient and you may need more elution strength. (or less lipophilic stationary phase).

First thing to try may be to let the gradient go up to 99% or 100% B.

If that is not working, maybe you can add something with higher elution strength on reversed phase. Maybe add some % of isopropanol to the acetonitril (never tried but; watch the pressure as IPA is more viscous. To some extend, even ethyl acetate may be possible, as long as it is miscible with the aquoues solvent).

On the other hand, if all your peaks elute that late in the gradient, you may start at higher %B (e.g. 50->95 or 75->95) which makes the gradient more shallow. This may also help.

If the peaks are already widely distributed over your gradient, then you probably already have your answer and isocratic is not the way to go (which then leaves you still with the same issue on the gradient separation).

thank you for you response.
when I said "after the gradient" I meant the 20 minutes i had set in the method. my last peak came off at 22.3 minutes. is there an easy way to find what the gradient delay would be?

all my peaks were coming off after 15 mins (initial excitement coz that would mean isocratic would work as all peaks within <25% of the gradient time, until i saw the peaks coming off beyond 20 minutes). I repeated my gradient going from 50%-100% acetonitrile which improved it slightly but my last peak still came off at 21.1 minutes. also this spread my peaks out over the whole 20+ minutes, not sure if this will mean my sample is not suitable for an isocratic method.

thank you for you response.
when I said "after the gradient" I meant the 20 minutes i had set in the method. my last peak came off at 22.3 minutes. is there an easy way to find what the gradient delay would be?

Have a look here or at the PhEur.
https://www.lcresources.com/resources/TSWiz/hs410.htm

In slight adaption, I like to do the gradient from 5->95 and also add some 3-5 min of isocratic steps before and after the gradient, just to get steady baselines of the signals (of course that isocratic step has to be subtracted when calculating the result).