I am trying to develop a HPLC method for the quantification of carotenoids using a C30 column that was bought recently but I am running into some issues.
I do not see a peak for the analyte (beta-carotene). However, a peak always appears after 3 minutes for all the conditions tested and no other peak appears throughout the test duration as seen in the picture. Please what could possibly cause this?
Column: C30 100A 3μm 150x4,6mm
Detector: JASCO MD-2015 Plus diode array detector
Analyte tested: beta-carotene standard (20 µg/ml) prepared using acetone as a diluent
Wavelength: 200 to 700 nm
Injection volume: 20 µl
Mobile phase: Methanol:Ultrapure water (98%:2%) (A) and MTBE (100%) (B)
Gradients and conditions tested
Thank you for your help and suggestions
Try looking online at Agilent.com , Waters.com , Phenomenex.com , Restek.com . But I think the concentration of your standard is too low! Check the USP and AOAC methods and use their concentrations. I think I could detect (it's been >50 years) 1.0 IU.
For sure, the peak at 3 min is not your analyte, most likely the analyte was not eluted in all the experiments , so before trying what I will suggest, clean your column according to the manufacturer's instruction.
Your compound is lipophilic logP is 9.7 (ALOGPS), so you have to use solvents with more elution power than methanol. you can also try increasing the flow rate as the internal diameter for your column is 4.6mm
Dear all. I have made some changes to the mobile phase (I used tbme,ACN and MeOH) and my analytes now elute. Thank very much for your contributions
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