I have done TLC of my plant extract and done spectral analysis in HPTLC instrument. I have checked spectra of my sample and compared with standard rutin sample which showed 2 peaks one at 254nm and another at 356nm but the former peak was broader and hence instrument showed 254nm as lambda max for standard. Whereas for my sample broader peak appeared around 362nm so instrument detected lambda max at that wavelength.
In literature lambda max of Rutin is given as 2 wavelengths i.e. 257nm and 359nm. So on what wavelength should I quantify Rutin?
It is not rare when compounds have several bands in UV spectra, corresponding to different excitation processes, for example pi-pi* and n-pi* transitions. For quantitation people normally choose lambda max with greatest epsilon value (to maximize sensitivity) unless it is obscured by UV bands of impurities. In this case it is vise to make quantitative method based on less intense but non-obscured absorption band.
Rutin is a phenolic compound and the phenolic groups, being acidic, may convert to their conjugate bases at various pH. So you may need to adjust the pH (or buffer your solution and mobile phase) to get consistent spectra. As mentioned above, also look for impurities in your extract.
Although this paper discusses fluorescence (and has AU on a scale where it should be emission), the fluorescence changes as a function of pH; to have fluorescence, one needs to have absorbance, so the changes in flourescence may be from changes in absorption at the excitation wavelength: https://www.researchgate.net/publication/232905721_Determination_of_Rutin_with_UV-Vis_Spectrophotometric_and_Laser-Induced_Fluorimetric_Detections_Using_a_Non-Scanning_Spectrometer
Figure 5 of this link also shows a change in absorbance at pH 4 and 9 (look for the 0 DPBA traces):
https://www.researchgate.net/publication/225962493_Modes_of_complexation_of_rutin_with_the_flavonoid_reagent_diphenylborinic_acid_2-aminoethyl_ester/figures?lo=1
Article Determination of Rutin with UV-Vis Spectrophotometric and La...
Article Modes of complexation of rutin with the flavonoid reagent di...
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Pharmaceutical Botany
- Plant Extracts