The presentation describes the principle and instrumentation of spectrofluotometer

1. SPECTROFLUORIMETRY
DR. SMITHA K. R.
ASSISTANT PROFESSOR,
DEPARTMENT OF BIOCHEMISTRY,
SREE KRISHNA COLLEGE,
GURUVAYUR

2. SPECTROFLUORIMETRY
Principle
• Fluorescence is an emission phenomenon, the energy
transition from a higher to lower state within the molecule
concerned being measured by the detection of this
emitted radiation rather than the absorption.
•

3. ➢ In order for the transition from higher to lower
states to occur, an earlier excitation event, for
example caused by absorption of
electromagnetic radiation, must have taken
place.
➢ The wavelength(s) of absorbed radiation must
be at lower values (higher energy) than the
emitted (fluoresced) wavelength.

4. ➢ The difference between these two wavelengths
is known as the Stokes shift and in general the
best results are obtained from compounds
involving large shifts.
➢ It is possible for a compound to absorb (be
excited) in the ultraviolet region and emit or
fluoresce in the visible region

5. • Most electrons will occupy the ground state S0V0 at room
temperature
• Elevation to a higher energy level, S1, S2, etc., may be
achieved by absorption of electromagnetic energy
(photons) in less than 10-15 s
• Energy may be lost very rapidly (as heat) by collision
degradation, resulting in minimal vibrational energy in
the lowest excited state, S1V0
• Electrons in this state return to the ground state in less
than 10 -8 s, the emitted energy being manifested as
fluorescence.
•

6. • Many organic molecules absorb in the
ultraviolet/visible regions but do not fluoresce
• The emitted radiation appears as band spectra
because there are many closely related values
(for the wavelengths) dependent upon the final
vibrational and rotational energy levels attained.

7. • One radiative mechanism by which excited
electrons may relax is a light-emitting transition
from the lowest excited state (S1) to ground
state (S0) in a fast (10-9 to 10-6 sec) process
called fluorescence
• The energy difference is dissipated by emitting
a photon
• Due to the electron having shed some of the
original excitation energy by vibrational
relaxation, the emitted photon will be of lower
energy and thus of longer wavelength.

8. Adapted from
https://www.enzolifescien
ces.com/sciencecenter/tec
hnotes/2019/december/
what-is-the-difference-
between-fluorescence-
phosphorescence-and-
luminescence

9. • These band spectra are usually independent of the
wavelength of the exciting radiation and have a mirror
image relationship with the absorption peak with the
greatest wavelength.
• An associated phenomenon is phosphorescence, but this
emission has long decay times and usually persists when
the exciting energy is no longer applied.

10. • Fast (10-11 to 10-6 sec) Intersystem crossing from singlet
exited state (S1) to an energetically favorable triplet
excited state (T1) leads to inversion of the electron spin
• Triplet excited states are characterized by parallel spin
of both electrons and are metastable
• Relaxation occurs via phosphorescence, which results in
another flip of the electron spin and the emittance of a
photon

11. • The return to relaxed singlet ground state (S0) might
occur after considerable delay (10-3 to >100 sec)
• Additionally, more energy is dissipated by non-radiative
processes during phosphorescent relaxation than in
fluorescence
• The energy difference between the absorbed and
emitted photon is bigger and the wavelength shift more
pronounced
• Thus, phosphorescence is characterized by a bigger
Stokes shift than fluorescence.

12. Adapted from
https://www.enzolifescien
ces.com/sciencecenter/tec
hnotes/2019/december/
what-is-the-difference-
between-fluorescence-
phosphorescence-and-
luminescence

13. • Phosphorescence arises as a result of intersystem crossing to
the lowest triplet state. This light emission usually occurs at
longer wavelengths than does fluorescence.
• Fluorescence spectra give information about events that occur
in less than 10 -8 s.
The ratio
gives Q as the quantum efficiency.
At low concentrations, the intensity of fluorescence (If) is related
to the intensity of the incident radiation (I0) by:
where c is the concentration of the fluorescing solution (molar), d
is the light path in fluorescing solution (cm), and ε is the molar
extinction coefficient for the absorbing material at wavelength
λ (dm3mol-1cm-1).

14. Advantages
➢ is most accurate at very low concentrations, whereas absorption
spectrophotometry is least accurate at these concentrations.
➢ increased sensitivity, which is easily adjustable over a large
range by amplification of the detector signal.
➢ Although no reference cuvette is required, a calibration curve
must be obtained.

15. 4. Instrumentation
❖Two Monochromators may be employed, the first (M1)
for selecting the excitation wavelength
❖Fluorescence emission occurs in all possible directions
and one direction (90°) is chosen and the second
monochromator (M2) is used for determination of the
fluorescence spectrum

16. ….Instrumentation
❖The radiation source is generally either a mercury lamp
or a xenon arc, excitation wavelengths frequently being
selected in the ultraviolet region and the emission
wavelengths in the visible region. The detector is usually
a sensitive photocell, for example a red-sensitive
photomultiplier for wavelengths greater than 500 nm.
❖Temperature control is required for accurate work as the
intensity of fluorescence may vary between 10% and
50% for a 10 0C change at approximately 25°C.

17. Two approaches
➢Simplest is the basic 90° illumination
➢front-face illumination which obviates
pre-and post filter effects

18. ✓ front-face illumination effects arise owing to the
absorption of radiation prior to it reaching the
fluorescent molecules (pre filter absorption) and the
reduction in the amount of emitted radiation escaping
from the cuvette (post filter effects).
✓ Such effects are more evident in concentrated solutions,
and the use of micro cuvettes (containing less material)
can be of value.

19. ✓FFI is essential for examining suspensions, and
cuvettes with only one optical face are required.
✓Excitation and emission occur at the same face but
generally the technique is somewhat less sensitive
than 90° illumination.

20. Principles and Techniques of Biochemistry and Molecular Biology edited by Keith Wilson and John Walker

21. Principles and Techniques of Biochemistry and Molecular Biology edited by Keith Wilson and John Walker

22. Applications
• Fluorescent probes
• Enzyme assays and kinetic analysis
• Protein structure
• Membrane structure
• Fluorescence immunoassay
• Fluorescence activated cell sorter

23. Reference
1. Principles and Techniques of Biochemistry and Molecular Biology edited by
Keith Wilson and John Walker; sixth edition: Cambridge University Press
(2005)
2. https://www.enzolifesciences.com/sciencecenter/technotes/2019/december/
what-is-the-difference-between-fluorescence-phosphorescence-and-
luminescence

24. Thank you