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Quantification of Riboflavin by

Fluorescence

Spectroscopy

Many molecules absorb in the UV-VIS radiation, making it

possible to generate UV-VIS spectra of them.

λmax

Some, emits the absorbed energy in the form of radiation ⇒

(fluorescence/ phosphorescence).

In general λmax of excitation (absorption) ≤ that of fluorescence (emission).

= λem λex =

Cy3

v=0,1,2..

radiative

Non-radiative

Non-radiative

HOMO

LUMO

ISC

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Fluorescence spectroscopy is a type of optical

spectroscopy which analyzes fluorescence from a

sample.

Amonochromator is an optical device that

transmits a selectable narrow band of wavelengths

of radiation chosen from a wider range of

wavelengths available at the input.

Greek: mono- "single", and chroma, "color“.

PfPin

S

Rmonochromators

Spectrofluorometer

1

2

• Source: Hg or Xe arc lamp.(continuous radiation, 250-600 nm).

• Monochromators (two): one to select radiation for excitation (UV-Vis) and the other to monitor emitted radiation from the excited analyte molecules (fluorophore) .

• Cells and Cell compartments: Usually rectangular in shape, 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)

Instrumentation \

Excitation radiation

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Emitted radiation

Signal =

λex ?

λex = λmax,UV-Vis

Riboflavin Vitamin B2

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

UV Absorption Spectrum of RiboflavinFluorescence Spectrum Generation

1. Generate the Emission spectrum:

Acquire emission spectrum by keeping the

exciting light at a constant wavelength (λλλλex, a high

energy photon) and measuring the different

frequencies of the fluorescence emitted by the

sample.

Use UV-VIS Spectrum as a guide to find a λλλλexa suitable excitation wavelength. (Usually

it is a λmax of the UV-VIS spectrum of the analyte).

λmax,UV

emissions

Determine the wavelength at maximum emission = λem .

Hold @

λmax,UV

Signal = emission spectrum

Fluorescence Spectrum Generation

2. Generate the Excitation spectrum:

Acquired by recording the fluorescent light emitted

at λem as the frequency of the monochromatic

incident (exciting) radiation is varied.

vary excitation λ

λ

Determine the excitation wavelength(s) that yields the

high emission(s) @ λem which is λex.

Hold @

λem

λex is close to λmax,UV.

In fluorimetry Pf,λem (power of fluorescent light)

measured for a fixed Pin,λex .

Pf,λem = 2.303 k’εex b C Pin,λex α C

Pf,λem,max = 2.303 k’εex,max b C Pin,λex,max = k C

Spectrum

Measure @

emission maxPf,λem,max = k C

Fluorescence Intensity, Emission Spectrum

Beer’s Law, Absorbance

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In fluorimetry, Pf is measured holding Pin constant so

that it is not necessary to determine Pin.

fP =k C

Fluorescence Intensity

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Fluorescence Spectrum of Riboflavin (Emission)

Pf,λem,max

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Fluorescence Spectrum of Riboflavin (Emission)

Pf,λem,max

Concentration of solutions ↑

kCPf=

Offset = ∆λ = λem- λex

λex λex

Excitation and emission monochromator setting

with the offset.

Synchronous spectra: Both monochromators scanned

but at a fixed wavelength difference (∆λ). Increased

discrimination of complex mixtures where fluorescence

is affected by a number of interferents.

P= k C

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Excitation, Emission and Synchronous Spectra

Peak height P = k C

Excitation, Emission and Synchronous Spectra

Synchronous spectra:

Low overlap, leads to a sharper maximum of the output signal.

P

Calibration curve

Acquiring Fluorescence Spectra:

Different types of fluorescence spectra:

Emission spectrum: Fix mono (1), scan mono (2). Usually regarded as

‘fluorescence spectrum’.

Excitation spectrum: Scan mono (1), fix mono (2). Essentially same as

absorption spectrum though relative peak heights are sometime altered.

Synchronous spectra: Both monochromators scanned but at a fixed

wavelength difference (∆λ). Increased discrimination of complex mixtures

where fluorescence is affected by a number of interferents.

Total Fluorescence: 3D spectrum. Emission spectrum scanned at

regular excitation intervals.

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 λmax value(s). With a reasonable value of UV λmax proceed to

determine a suitable λex and λem for the experiment** using the fluorimeter,

this is an iterative process.

Set up the parameters to collect the synchronous spectra with proper λex

and λem wavelengths. Keep other parameters at default values. Check the

[Detector]; S, R and S/R must be active.

Obtain the synchronous spectra of the standards. Obtain the synchronous

spectrum of the ‘unknown’ (triplicate). Calculate the mass (mg) of

Riboflavin in the tablet.