Chromatography Forum

Hello. I have just started with the simultaneous determination of vitamin A, D and E. I have tried the retention times for all of them and I am finding problems with vitamin A, as it does not appear in the chromatogram and I let it run for 40 min. I am using retinol palmitate and the working conditions are: 0,800 l/min, 280mV, 70%Hexane/30%Ethyl Acetate as mobil phase. Does anyone know where is the problem?

Thanks

I have run numerous Vit A tests and have used Mobile Phase: 95% wet hexane + 5% diethyl ether; flow rate 1 mil per min; for the UV setting please prep a 1:200 dilution of the stock retinol palmitate in wet hexane and measure the Absorbance on the UV to find out where the wavelength of maximum absorbance is such that you can set your LC Uv detector properly. I saw no need for any ISTD as the Vit A recovery is >95% and does well via ''external calibration".

Ok, I will try again. Sorry I made a mistake, of course the flow is 0,800 ml/min and 280 nm of wavelenght. Thank you very much for your answer

your hexane concentration is way too low. For vitamin A, I have found that using almost pure hexane achieves best results. What kind of column are you using? For vitamin A, we usually run at a wavelength of about 325 nm, using pure hexane. For Vitamin D, we usually run at 265 nm, using 95:5 Hexane:Ethyl Acetate

Hello! I tried with Hexane:Ethylacetate (95:5) and I have obtained a small peak for vitamin A for a concentration of 100 ppm. The area was about 5000, isn´t it too small for this concentration? The calibration curve I have to do is supose to be in the range 0,15-12 ppm, so I don´t know if for these concentrations a peak will be detected.
Thank you for answers