Help regarding baseline problem

[sathyavrathan]

I have problems in base line setting with HPLC software.
The baseline is not proper and gets shifted when a sample or standard is injected. Also, we are not able to get area of the peak from the chromatogram.
I have attached pictures of the chromatogram.

Please help in rectifying these problems and carryout the processes in place.

Instrument details:
Shimadzu UFLC20AD
Model: CTO-10ASVP

RID: RIDLIOA
Diode detector: SPD M20A
Software: LCsolution software

Expecting a reply at the earliest.

[danko]

Do you experiance the same baseline shape when you inject a blank sample i.e. solvent? If you havn't tried it, that would be an important action. As well as injecting nothing - i.e. zero injection.

[sathyavrathan]

when injecting blank, the baseline is straight. Zero injection is also fine.
But about 5 minutes after injecting the sample or standard (after the 1st peak) there is unsteadiness in baseline as you see in the picture.
I am really conerned about this and donot know why its happening.
help me in sorting this out.

[danko]

OK. That means the samples contain something (s) that elutes in an uncontrolable manner i.e. doesn't form a peak or peaks. That something absorbs ligth at the working wavelemght. The phenomenon is often observed when ionized substances elute in pH uncontrolled mobile phase.
My suggestion: lower the mobile phase pH to something like 2.5 using a buffer. Could be phosphate. Or add some TFA to your current mobile phase.

Best regards

[Fernando]

Hi sathyavrathan

What is the mobile phase? In what solvent you dissolve your sample?

Best regards

Fernando

[sathyavrathan]

Thanks danko.
I am using 5mM H2SO4 as mobile phase and HPLC water to dissolve the sample.

Its all done as given in the literature.
what else may the cause for the problem??

[danko]

OK – not a buffer, but might be OK.
Please let me know:
1. What does the sample matrix consist of (some salt/buffer, or some polymer?)
2. What is the pH in the sample solution
3. What is the mobile phase organic modifier Acetonitrile, Methanol, Propanol….
4. Detector wavelength choice

Then we might find a solution to your problem.
Best Regards