Chromatography Forum

Dear Chromatographers,
We have problems when to develop a method. Here is the condition:
Column 125 x 4 mm, C8 (5 um), mobile phase: meOH : phosphate buffer 0.02M pH 7.00 contains 0.1% TEA (65:35), detection 260 nm, flow rate 1 ml/min, oven temp 40 C.
With this cond we can separate the main peak with impurities (1 small peak in front - about 8.5 min, 2 small peaks behind).

But, we got something odd with the baseline:
About 4.5 min the baseline increased sharply (red arrow) and then downward at a snail's pace to create a slope.

The questions are:
What is that? What kind of substance that could create the phenomenon? How to overcome the problem?



Thank you for every information and suggestion.
Best regards,
SYX

Without being able to find your chromatogram, my guess would be that you got an air bubble in the detector cell (maybe even somewhere else) which slowly dissolved away.

I think I have another problem here...
Why the picture does not show up?

I will describe our chromatogram here... (I need help to show it).
The odd "peak" show up on every chromatogram at about 4.5 min and then gradually back to normal baseline after 18 min. our main peak stands on the slope (11.5 min) and difficult to integrate due to the slope.
When we tried to change mobile phase composition, the retention of odd "peak" will be change.
If it is bubble, as Mr. Muller said, from where it comes from? Injection port? I think not, I did not find the same trouble with other samples with different component.
If it is an impurity, what kind of component that can create that shape of peak in RP-column?

Exactly what are you injecting?

Could be a sample matrix issue or you may be at a pH were you have partial dissociation of component. My guess is you have an ionic species in there somewhere that isn't happy at pH7 or the sample pH has not been adjusted to match the mobile phase. For instance, injecting a sample at pH 1 onto a system running at pH 7 can cause this sort of problem. Need to know more about the sample.

The TEA present would make me suspect that you are working with bases, and possibly the pH may be too low. Double check your pH and determine if the method is written for actual pH or appearant pH

Make sure sample is prepared in mobile phase and that the pH of the sample you are injecting is the same as the mobile phase.

I wrote "pH" 10 times, so I guess that is where I would start investigating.

We injected active substance solution (not pure standard solution). It may contain impurities.
The substance has piperidine ring in its structure. pKa values: 8.91.
Our condition is OK for the “main peakâ€

What does an injection of a blank (H2O) look like? Could well be an inj. incompatibility as mentioned, though I wouldn´t give up air either, yet.

Syx...I think you are on the right track. If you're not using an RP column that will handle high pH, get one, there are lots out there. You can also try collecting your unknown peak as it elutes and getting a mass on it. That might help clue you in to what you have.

If your peak shape improves at pH10 than you can either run the method at elevated pH or go to the appropriate Primesep** column with it's mixed mode capability and milder conditions.

**I hope I don't get hammered for this....

** Careful, big brother is watching

I can show you the chromatogram!
Mr Mulller, I never inject water to the LC system, but I have injected diluent and found no interferences as in sample solution.

How clean is your mobile phase? This looks nearly like a displacement train, although it is hard to imagine for soemthing like that to happen in normal chromatography.

One picture is certainly worth many words.

Syx, you said that the retention of the baseline shift changes with mobile phase composition. Is the change "typical" of reversed-phase (i.e., is the % change in retention about the same as that of your main peak?). If it is, that's an argument for something in your sample that simply tails horribly (along the lines of Supercritical's suggestion).

The relative retention time is same in different solvent strength. What should I do if it is really a super-tailing component?
If I need to use pH 10 or higher I should change some spare-parts those are not alkaline-resistant.
Mr. Neuwe, we use bidestillated-demineralized water, other components are in HPLC grade. We always filter the mobile phase using membrane filter with pores 0.45 um.

Ok, seeing the chrom now: It really looks like what I have seen, sometimes, when air was co-injected, though I don´t understand why your blank doesn´t do this (why don´t you inject H2O?). Do you concentrate the samples with a stream of gas?