injection volume is changing the chromatography

[R&D-Chemist]

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

[Consumer Products Guy]

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

[WillyOne]

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

[JGK]

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.

[R&D-Chemist]

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