I run a compound on HLPC at four different wavelengths (205,215, 254, and 306nm), and my solvents are 30% IPA and 70% Hexane. Absorbance peaks are shown at the same time in those wavelengths. However, the intensity of those peaks are different, and one of peaks disappears in one of the four wavelengths. My questions are how to choose best wavelengths to determine %ee and whether or not I should stay consistent at one wavelength ( because one of the peaks disappeared. It is almost a straight line on a spectrum).
I am new to HPLC. Please help me explain more, and all comments are appreciated.
- "Proper Wavelength Selection for HPLC Method Development (or Purity Determination)"; https://hplctips.blogspot.com/2013/08/wavelength-selection-for-method.html
If you have multiple compounds present in the sample and an HPLC scanning diode array detector inline for analysis, then select the appropriate best lambda max signal for EACH sample. Make sure to have your teacher help you use the HPLC system.
"How to analyze HPLC spectra at different wavelengths ?" ??? Your question needs to be rephrased.
- "Terminology is very important in science. "SPECTRA" include a range of data / discreet wavelengths scanned across a defined range of wavelengths (e.g. Spectral data were collected from 200 to 400 nm). This provides a spectral plt (3D) which can be viewed for determination of which wavelengths a compound(s) shows absorbance at.
- Wavelengths are discrete values (e.g. 280nm, 10. *280nm with a 10nm bandwidth). Some types of UV/VIS detectors can collect multiple wavelengths that YOU specify in the software (i.e. 210, 230, 254nm). Some types of scanning detectors can collect multiple, discreet wavelengths AND also collect and store full spectra over time or across a sample peak (e.g. Spectral data were collected from 200 to 400 nm) which is a very useful feature for method development.
- "Modern HPLC Method Development Tips (PART I)"; https://hplctips.blogspot.com/2016/07/modern-hplc-method-development-tips.html
- "Modern HPLC Method Development Tips (PART II)"; https://hplctips.blogspot.com/2016/08/modern-hplc-method-development-tips.html
You should not be surprised! Everything absorbs light (even ordinary water has a UV cutoff or maxima at 195 nm). Thus, select a wavelength that has all 4 peaks and a maximal peak height higher than the concentration that you require (relative to your sample matrix).