2 Replies Latest reply on Feb 23, 2019 7:41 AM by saragalal

    Narrow stokes shift

    saragalal

      I am analysing two compounds which show an overlap between the higher wavelength end of the excitation spectrum and the lower wavelength end of the emission spectrum (one compound with excitation wavelength at 280 nm and emission wavelength at 300 nm) , the other one with excitation wavelength at 290 nm and emission wavelength at 304 nm

      i have two questions:

      1- can these emission wavelengths be used for compounds quantification in presence of this overlap (risk of interference between excitation and emission wavelengths at the detector)??

      2- if not , what is the instrumental manipulation that can be done to solve this problem to achieve better separation between the two spectra( excitation and emission)??

        • Re: Narrow stokes shift
          Mike Picollelli

          Hello saragalal;

           

          I apologize for the delay in answering your question.  I hope the answer helps.

           

          1- can these emission wavelengths be used for compounds quantification in presence of this overlap (risk of interference between excitation and emission wavelengths at the detector)??

           

           

          These wavelengths (i.e. 280/290 nm and 300/304 nm) are indeed very close. With the broad nature of most fluorescence bands, I believe there will always be some level of overlap. This can be minimized by selecting a very low SBW (Spectral Bandwidth) value. Lowering the SBW will improve resolution of the scans, however Signal to Noise ratio will suffer slightly.

           

           

           

          2- if not , what is the instrumental manipulation that can be done to solve this problem to achieve better separation between the two spectra( excitation and emission)??

           

          Lowering the SBW values can improve resolution and separation of the peaks, assuming the peaks are natively narrow enough to be resolved. Many peaks stemming from fluorescence events are quite broad by nature.

           

          Best regards,

           

          Mike

            • Re: Narrow stokes shift
              saragalal

              Dear mr/ Mike Picollelli

              thank you for your reply

              i already used excitation  and emission bandwidth of 5 which i think is enough to exert certain spectra resolution but unfortunately got the previously discussed narrow stokes shift

              so i tried different excitation wavelength even if it would little compromise  sensitivity and already got satisfactory stokes shift but due to the very close structures of the two compounds, upon exciting the two compounds with wavelength 210 nm , i got the same emission wavelength (about 280 nm) for both compounds, then i tried to separate them in synchronous mode and succeeded to separate each one individually by first derivative synchronous and got intersects in which i could quantify each one without interference from the other

              finally i tried the same synchronous parameters but on the mixture of the both compounds together in the same solution. unfortunately the two spectra overlaid in the mixture and to this point i have no solution

               

              my last question is the exact role of PMT voltage and when to use this option???

               

              thanks again. wait for your help and feedback,

              sara