I am running estradiol (alpha and beta), which have been dissolved in MeOH and stored in glass vials @-20C, on HPLC using an Agilent Zorbax Eclipse plus C18 column. I have run a standard of 1000 ng/mL under the same conditions 3x, and I am getting inconsistent absorbance (as low as 5 mAU and as much as 100 mAU). What could cause such widely different absorbance? The samples are brought to room temp, agitated, and the sample is taken from the bottom of the vial. I am trying to be consistent but not getting consistent results. Is there something about estrogen in alcohol that I am missing? Suggestions?
I had no problem in the past with Estradiol Valerate (an ester). I used 80:20 Acetonitrile:Water as the mobile phase. Try it!
I also used amber glass vials.
Dear Bruce.
Thank you for the response on the said question.
i am having a similar problem on mine.
I am using Agilent 1100 HPLC system for detection of pregnanolone. its dissolved in chloroform as instructed by the manufacturer (Santa Cruz). Mobile phase is ACN/ Distilled water 80/`20. C18 reverse phase column. pressure at and flow rate at 1mL/minute. some peaks appear at
Yes. Normally, a 5 cm HPLC column flowing at 1.0 mL/minute will have a molecule elute between 5-10 minutes. A 250 cm column flowing at 0.5 mL/minute will have a molecule elute at 10-15 minutes. But this is not a general rule as long as you stay well away from the injection front.
Thank you Bruce N. for your prompt response.
''But this is not a general rule as long as you stay well away from the injection front.''. What does that mean?
Thank you
Elution times vary based on many parameters, but 'real chromatography (were the capacity factor is between 2-10)' occurs at those times. I certainly would not tear my hair out if the molecule elutes at
Many thanks Bruce.
i got so much help from you this day.
Regards
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