HPLC noisy baseline

[sgkhayade]

With Sodium orthophosphate buffer 0.01 M +Heptane sulphonic acid 2gm/lit adjusted pH 2.5 , Baseline is very noisy after 20 min at which time Acetonitrile is 75% in gradient.Please explain the reason.

[zokitano]

Have you tried to stabilize your column with the mobile phase longer, to ensure less noise during the runs?

[danko]

Hi Santosh,

I’m not sure, I have the explanation yet, but I’ve experienced a similar problem.
My observation was made on an Acquity system and the noise appears only in gradient mode. The isocratic baseline was a little bit noisy, but without any changes/deviations. When a gradient was set, the noise appeared at certain (high) ACN concentrations and in a very reproducible manner.
What kind of hardware (HPLC brand and model) are you using? Does the noise disappear when 75 % ACN is exceeded? Did you try isocratic runs with 75 % ACN concentration and with another (reference) concentration? I assume you do not inject any sample.
My observation was made both with phosphate and TFA based mobile phases, so I don’t think this is specific to a certain chemical.

By the way: Isn’t pH of 2.5 somewhat lower than it should be bearing in mind that the sulphonic acid should be dissociated in order to act as an ion pair? I can’t find Heptane sulphonic acid’s pK value but I would expect it to be above 4. You could have some solubility issues there.

Best Regards

[juddc]

Running gradients with ion pairing agents is tricky business. In general it takes a long time for systems with IP agents to come to equilibrium because a certain portion of the IP agent actually is retained on the column, I believe, and running a gradient upsets that equilibrium. One might expect not only a noisy baseline but also possibly irreproducible chromatography. I'd suggest using an isocratic method if at all possible or finding chromatography conditions that do not require the use of an IP agent. It's a lot of work, I know, but I see lots of pain ahead in working with a gradient using an IP agent.

Maybe (and it's a very thin maybe - so thin that I hesitate to bring it up here for fear of being strung up for being stupid) if you dope the organic portion of your MP with a heavier clip of IP agent, you might quiet things down a little by keeping roughly the same amount of IP agent on the column over the course of your run. Check some of the charts in Snyder's Practical HPLC Method Development for guidance on IP agent concentration at different MP organic levels. I wouldn't bet on it though if you manage it, I'd like to hear about it.

Running at pH 2.5 should be fine. I believe the pKa of most commonly used aliphatic sulfonates is ~1.5 or so.

Check this from the archives:

http://www.lcresources.com/discus/messa ... 20041231pm

[danko]

Hi juddc,

Thanks for pK info. I wonder why it’s not listed along other properties of Heptane Sulphonic acid?

Juddc wrote:

Running gradients with ion pairing agents is tricky business. In general it takes a long time for systems with IP agents to come to equilibrium because a certain portion of the IP agent actually is retained on the column, I believe, and running a gradient upsets that equilibrium. One might expect not only a noisy baseline but also possibly irreproducible chromatography.

As I wrote, my story was based on two different mobile phase systems: TFA is the one and the other contains no ion pair reagent at all (phosphate buffer + ACN)
Another thing you might have missed is that the noise appearance followed by disappearance is very reproducible. When the gradient was run twice, the noise would appear roughly at the same time and for the same duration.

What I forgot to mention is that the same gradient was run with the same mobile phases on another HPLC model (Waters Alliance) and the noise was not observed. So my feeling is that we are dealing with a hardware problem of some kind.

Juddc wrote:

I'd suggest using an isocratic method if at all possible or finding chromatography conditions that do not require the use of an IP agent. It's a lot of work, I know, but I see lots of pain ahead in working with a gradient using an IP agent.

We should remember that TFA is an IP reagent too

Best Regards

[Serg]

do you have proper degasing setup? mixing water and ACN might produce a lot of bubbles and might affect your baseline.

Regards.
Serg

[sgkhayade]

Dear friends
Thanks for your suggestions.
This is very specific problem I faced.
The noise is not reproducible at each run.
I tried after stabilising the baseline for many hours.Then also the problem persists.
I have tried different types of best quality reagents.
The baseline is noisy; not due to the reason that I am using excess amount of IP reagent.Because the same gradient I am using for different product but at pH 4.0.
Finaly I got the solution!!!!!!!!!
When I was filtering the buffer taht time it was reacting with filter paper and degaded product was eluting in the run.When I tried without filtration I got very good baseline throughout the run.
I conformed it by using Different brand filter paper at which time also i got straight baseline.

[tom jupille]

Santosh, thank your for getting back to us with the solution. It helps us all to learn!