Chromatography Forum

Hi,
I need your help to explain this phenomenon:
Mobile phase A: 75:25:water:acetonitrile, 0.1% Formic acid
Mobile phase B: 5:95: water: acetonitrile, 0.1% Formic acid
Column:Waters Symmetry C18 4.6mm x 150mm, 3.5um
Gradient: 100% mobile phase A to 100% mobile phase B in 25 minutes.
Symmetrical Peak shape C detected in blank run (20:80 water; acn), area = 10.
Let system run idle for 2 hour and then make blank injection, peak C looks like a square, peak area = 300. Subsequent injections show peak C looks normal, peak is symmetrical, area = 10.
Let system run idle again after 1.5 hour elapsed interval and then inject blank, peak C looks like a square again, area = 290. Inject another blank right after this and peak C looks normal, area = 12.
I repeat another one and the result is the same: abnormal peak shape and high area count.
What causes these abnormal peak shape and area count when the sequence injections are not made quickly in succession?
Thanks in advance for your help.

It looks to me that you have a significant flow rate problem: when you let the system run for some time, you have pump failures. When your peak elutes, the pump stops (or runs at low flow) for some time, which gives you the square peak with the large peak area.

Hi, Uwe,
The flow rate was normal as there was no shift in retention time of the main peak and area count. Peak C (found in the diluent) was the only one affected. I ran another method validation on this LC and it works fine.

Does it happen with a new column?

Have you tried to use a freshly prepared mobile phase ?
Maybe you are concentrating impurities of the solvent on the column?

Hi, bhuvfe,
The mobile phases were freshly prepared each time. I repeated the experiment a week later using the same column and it yielded the same result (abnormal peak shape and high area count observed on peak C).
As you have suggested, the chance for impurities of the solvent to accumulate on the column seems applicable in this case because the area count of peak C increased with increasing time elapsed.
Thanks,

Sounds like classic building up of some contaminant from the water used to prepare the MP. Try prefiltering the water through an Empore extraction disk before making your A phase or (if you have a high pressure mixing, 2 pump system) put a C18 guard column between the A-phase pump and mixing chamber.

Thank you, Dr.
I will switch the water source to see if the peak C area scales up during equilibration.

I am still for the flow rate problem, and I am basing this primarily on the description that you got a square-shaped peak with a much larger peak area than the peak that eluted at about the same time. Here is my reasoning: You are running a gradient; your retention is not expected to shift, if your pump is working intermittently, but you can still get flat peaks, if your flow stops in between sometimes, especially when the peak is eluting.

One can get square-shaped peak also when it is big enough to overload the detector (e.g. too high absorption in UV detector) or be cut by the software (I had this software problem in GC separation even though my peaks were not overloading the detector !). However, the peaks were not ideally square-shaped but only cut on the top. Perhaps this in not a pump problem.

Ntroung,

Some chromatograms might help tremendously – I think.

Best Regards

Thanks to you all for your help.
I don't know how to post a chromatogram. Please guide me.
Thanks,

Follow this link to find the guide.

viewtopic.php?t=2617

Best Regards