4/25/2013

Fluorescence spectroscopy is a type of optical spectroscopy which analyzes fluorescence from a sample.

Quantification of Riboflavin by Fluorescence Spectroscopy

Molecular Luminescence
Certain molecules absorb photons; electrons promoted to excited states is followed by relaxation. Relaxation processes converts the molecule back to the ground state: Radiative relaxation luminescence Forms of photoluminescence (luminescence after absorption) are fluorescence (short lifetime of S1, S1 to S0) and phosphorescence (long lifetime T1, T1 to S0). Emitted light is used to determine certain properties (structure and concentration) of the emitting species. Very sensitive technique: detection limit - ppb Limited number of systems photo-luminesce.

Non-radiative

LUMO
ISC

radiative

HOMO
v=0,1,2.. Non-radiative

ex

Non-radiative

LUMO
ISC

radiative

HOMO
v=0,1,2.. Non-radiative

4/25/2013

em

Cy3

max of excitation (absorption) that of fluorescence (emission).

Spectrofluorometer
S Pin
monochromators

Instrumentation Source: Hg or Xe arc lamp.(continuous radiation, 250-600 nm). Monochromators (two): selects both wavelengths, for excitation and emission. Can focus on narrow absorption or emission peaks. Cells and Cell compartments: Usually rectangular all four sides transparent; quartz or glass depending upon the wavelength range needed; Radiation from fluorophore at 90 to the direction of incident beam is detected. Detector: Photomultiplier (amplifies small signals)

Pf R

ex em

Riboflavin Vitamin B2

4/25/2013

UV Spectrum of Riboflavin

Fluorescence 1. Excitation spectrum: is measured by recording the fluorescent light emitted at a frequency (em) as the frequency of the monochromatic incident light is varied. (~UV-VIS absorption spectrum) or UV-VIS Spectrum would be a start to find ex. 2. Emission spectrum: is measured by keeping the exciting light at a constant wavelength (ex); high absorbing and measuring the different frequencies of fluorescent light emitted by a sample.

http://www.pmda.go.jp/english/pharmacopoeia/pdf/sixteenth_edition/JP16%20UV%20P%20to%20Z.pdf

Fluorescence Intensity, Emission Spectrum In fluorimetry, Pf,em is measured for a fixed Pin,ex so that it is not necessary to determine Pin,ex i.e. only one measurement of light intensity is made, Pf,em . Spectrum Pf,em = 2.303 kbC Pin,ex,max C

Fluorescence Spectrum of Riboflavin (Emission) Pf,em,max

Measure @ Pf,em,max = 2.303 kbC Pin,ex,max = k C emission max

http://www.vernier.com/innovate/monitoring-vitamin-b2-in-energy-drinks/?utm_campaign=130205newsletter&utm_medium=email&utm_source=cl-customers&utm_content=html

Excitation, Emission and Synchronous Spectra

4/25/2013

Excitation, Emission and Synchronous Spectra

A stock solution (ca. 100 uM) Vitamin B2 in 1% acetic acid is provided. Make six standards, from ~10uM through ~80uM; make sure to use 1% acetic acid for diluting the stock solution. Prepare the most concentrated standard first and the other standards later. Prepare the unknown sample by dissolving the tablet in 10mL 1% acetic acid in a 50mL beaker. Filter quantitatively into a 100mL volumetric flak. Dilute to mark with 1% acetic acid. Take the UV spectrum of the 100uM solution from 200-600nm. Determine the UV max (ex) values. With a reasonable value for max proceed to determine a suitable ex and em for the experiment** using the fluorimeter, this is an iterative process. Set up the parameters to collect the synchronous spectra with proper ex and em wavelengths. Keep other parameters as default values. Check the [Detector]; both S and R must be active.

Synchronous spectra: Low overlap, because of the sharpness of the signal. Peak height = k C