Spectra This page summarizes the optical absorption and emission data of Riboflavin that is available in the PhotochemCAD packageversion 2. I reworked their data to produce these interactive graphs and to provide direct links to text files containing the raw and manipulated data. Although I have tried to be careful, I may have introduced some errors; the cautious user is advised to compare these results with the original sources.
When a molecule absorbs a photon in the ultraviolet or visible UV VIS region - nman electronic transition occurs within the molecule. This transition involves moving an electron from the singlet ground state to a singlet excited state.
Theory An excited molecule will undergo de-excitation to regain its ground state electronic configuration. De-excitation of the molecule can occur in three distinct ways: The first relaxation mechanism, collisional deactivation, occurs when the excited molecule transfers its excess energy to molecules with which it collides without photon emission.
The second mechanism, fluorescence, involves the electron returning from the excited singlet state to the ground state accompanied by the emission of a photon of lower energy longer wavelength than the absorbed photon; the energy loss is due to vibrational relaxation while in the excited state.
When fluorescence is favored, it occurs within about seconds after absorption picoseconds are seconds. UCD Fluorometer Phosphorescence, the third route for de-excitation, occurs when the excited electron enters the lowest triplet state from the excited singlet state by intersystem crossing, and subsequently emits a photon in returning from the lowest triplet state to the singlet ground state.
These singlet-triplet and triplet-singlet transitions involve electron spin reversal, which is a low probability occurrence. Thus, the time between absorption and phosphorescence can be from seconds to several minutes. Pathways for production and de-excitation of an excited state.
Since our interest involves fluorescence, a typical experimental arrangement is shown in Figure 4. This is used in either of two distinct ways. The first, which holds the excitation monochrometer M1 fixed, and varies the emission monochrometer M2, yields an emission spectrum the wavelength distribution of light emitted by the excited singlet state.
Alternately, M2 can be fixed and M1 varied; this procedure produces an excitation spectrum a plot of fluorescence intensity as a function of excitation wavelength. Often, the excitation spectrum of a pure compound has exactly the same profile as the absorption spectrum. Schematic Diagram of Fluorometer.
Coupling the above techniques with a relationship between fluorescence intensity and concentration would be exceptionally useful. The fraction of absorbed light is: Its two predominant irradiation decomposition products are lumichrome and lumiflavin, which are themselves highly fluorescent.
Riboflavin A and the two most predominant irradiation decomposition products, lumiflavin B and lumichrome C. The analysis method requires working with riboflavin samples at concentrations well below the part per million ppm level.
Therefore, it is extremely important that the utmost care be exercised in cleaning and handling all of the glassware and solutions during this experiment.
In the first lab period the standard and unknown solutions mentioned below should be prepared. Later during this period you should consult the T. You should also obtain the absorption spectra of riboflavin and perform a practice run on the fluorescence spectrometer. Deliver 1 mL of the stock solution to a 10 mL volumetric flask and dilute using deionized water.
This is your 10 ppm solution. Using a volumetric pipet, deliver 1.Riboflavin fluorescence is extremely sensitive to its environment. It is sensitive to pH, presence of oxidizing species, and exposure to light.
Measure three emission spectra; use excitation wavelengths of , , and nm and start emission scans at , and nm, respectively.
Fluorescence Intensity vs Concentration of Riboflavin The fluorescence intensity vs concentration graph of riboflavin above was created by diluting a stock solution of 50ppb, to 40, 30, 20 and 10ppb and measuring it in the fluorescence instrument.
The fluorescence emission spectrum of Riboflavin dissolved in ethanol. The excitation wavelength was nm. The quantum yield of this molecule is (Koziol, ; Sun, ). Once the fluorescence spectrum was measured the peak of the spectrum was found, and using the wavelength value that the peak in the fluorescence spectrum occurred at and a scan range of nmnm, the excitation spectra could then be measured.
The fluorescence intensity vs concentration graph of riboflavin above was created by diluting a stock solution of 50ppb, to 40, 30, 20 and 10ppb and measuring it in the fluorescence instrument.
As can be seen in figure 2, as the concentration of riboflavin increases, so does the fluorescence intensity, and the graph is linear, in the form y=mx+c. Fluorescence Spectrophotometry of Riboflavin By: Andrew Tam.
Introduction. Also, the spectra differ at fluorescence values because they are all at different concentrations of riboflavin. Chemisty / Natural Sciences pm, April 1, 1. Fluorescence Riboflavin spectrophotometry.