Chromatography Forum

Hello people,
can you advise me?
Is every raise of the baseline during gradient (increasing organic eluent), due to contaminents of the aqueous eluent?

What happenes if both eluents are contaminated?

thank you
and may peace be upon you

K.Zanov

What do you think about a variation in the absorption coefficients. Some contaminants would produce peaks instead of a baseline drift.

nope i'm talking about MS baseline raise.
the more abrupt is the gradient the more abrupt is the raise.
and furthermore i have two "ghost peaks", after the raising starts.

In this case, take care of the better ionization in presence of organic solvent respect to the water.

i didnt' quite get you...
You mean perform a tuning?

No.
Every time, when you increase the organic content in your mobile phase respect to the aqueous part, you'll obtain a better ionization because of the increased evaporation "speed" of the solvent in your sample, both in case of infusion and in case of chromatography.

So you suggest a more gradual gradient and therefore a longer analysis?
Sory man but I still don't get what do you mean by "respect".

If you work with gradient RP-LC, regardless which gradient you use, you'll face with baseline drift.
This is the reason why it'd be preferable to find an isocratic step in your gradient elution if you want to increase the robustness of your integration method.
Respect:
85% H2O / 15% MeOH starting composition
15% H2O / 85% MeOH final composition
in this case I increased the organic content respect to the aqueous content.

thanks for the advice

You can generally speaking identify if the baseline raise is due to contaminants or what Sam is saying by changing the equilibration time. If baseline increase correlates with equilibration times then contamination might be your problem (as well).

So it is! My system is contaminated.
I used 3 different kinds of water and the problem still persists.
I got 2 ghost peacks.
I they are there even when I changed the column.
And yes, the more the equilibration time is, the bigger the ghost peaks are.
If Anyone has an idea where is it contaminated and how can I solve this, please reply.

Thank you
May peace be upon you.

Is your water of HPLC quality? Do you see the peaks when you re-equilibrate with 100% water (your impurities can also be in ACN if you starting conditions contain (low) percentage of ACN)... You can always filter your water with by passing it through a C18 column and then collecting it. If that solves the problem then probably your water is contaminated or of not an adequate purity...

good idea, Kostas, I'll try that!
thanx!

I tried 100%water (HPLC grade) - same situation, a filtered it through the column, the "ghosts" are still there - same retention times, increasing in intensity with the equilibration time.
So now I am convinced it is somewhere in the sistem (I excluded the tubings)
Anyone, experienced something similar? I am runing out of options here!
can it be the degasser, mixer, injector...

what do you think about the in-line filter for the phases?