Chromatography Forum

I have developed the HPLC method for both Assay/RS of finished product & API.
I have same method for Assay/RS except injection volumes are different. 10uL for assay method and 50uL for RS method.
Now I am having issues with RS chromatogram.

My previous run was fine with same conditions (includes API-RS & FP-RS).


Chromatographic Column:

Mobile Phase: Follow the gradient profile below.
Mobile phase A : phosphate buffer pH 2.5
Mobile phase B : combination Mobile phase A and ACN
Atlantis d-C18, 3μ 4.6 x 150 mm


Flow Rate: 1.0 mL/minute
Column Temperature: 30˚C
Detection Wavelength: 225nm
Run Time: 35 minutes
Injection Volume Assay Test – 10µL
Related Substance Test-50µL

Following are the results when injected same sample(same vial) at different injection volumes.
http://tinypic.com/r/35kv2gi/7





http://i55.tinypic.com/335cnz4.jpg

50 ul is a large injection. Especially if your solvent in your vial is higher organic level than your initial mobile phase.

It seems that your absorbances for the peak are also very high.

If your sample matrix has a high organic content, the 50 µL injection may be producing localised column property effects. For a 5 fold increase in injection you are not even seeing a two fold change in peak height.

Tailing at the front end of the peak (50µL inj) could ne the start of column overload.

In regular chromatography, I would attempt to keep all my peaks < 1 AU in height.

Thank you all for the responses.

And i will agree you all. I guess i have messed up with diluent preparation. I prepared new sample in less organic solvent and injected again. everything looks good.

Thanks again