I found that my HPLC fluorescence detector couldn’t quantify tocopherol 1000ppm while an UV detector can. Why does this happen? Is it that the fluorescence detector is too sensitive for higher level detections?
When I plot the external calibration curve, if the concentration of tocopherol standard is above 250ppm the peaks fall down to baseline (which shows system quantifying error) which only happens in fluorescence detector. Kindly advise.
You are saturating your detector. Dilute your sample, or use your UV data to quantify higher concentrations.
Is that means Fluoresence detector is not suitable for concentration more than 100ppm?
Yes. It is a trace level detector normally. At higher concentrations the calibration curves roll back over. For anything over, oh, around 100 ppm you should go to UV and if you need a second detector maybe ELSD.
Alpha-tocopherol quinol form has a strong native fluorescence in organic solvents with the fluorescence intensity and maximum emission wavelength dependent on the solvent used. You didn't mention the solvent you use but generally the concentration is too high and obviously the detector flow cell is saturated. If you don't wish to alter your experimental setup/change solvents then you need to dilute your solutions by a factor of 100. Even a 100 ppm is very high for a fluorescence detector in this case.
You can decrease the volume of injection was 1 mu.l and configure the low sensibility 1 and the gain may be you can reach this concentration.
And also to couple uv and fluorescent it's very better.
Flouresence detector is very much sensitivity compare to UV detector. When sample concentration is very PMT of FL Detector saturates and signal goes deep negative in from the baseline. If you want to upto 1000 ppm conc for linearity either use UV detector or decrease the injection volume while using FLD.
I have attached an application note for for separation of Tocopherols on UV detector.
Te estas enfrentando a una de las técnicas más rigurosas en HPLC. El tocoferol (vitamina E), es un compuesto de baja o nula solubilidad en compuestos polares; por esta razón debes intentar usar cromatografía en fase normal; que requiere de un poco de estrategias ya que un alto porcentaje de equipos HPLC se usan con fase reversa.
Es cierto que el detector de fluorescencia es más sensible que el UV; sin embargo esto es solo para la moléculas capaces de producir el fenómeno de excitación y emisión; y aquí hay que tener muy encuenta que los solventes usados no te estén quenchiando la señal
You can you methanol and acenitrile for good separation with reverse phase.
- Badges
- Science method