1. Molecular Luminescence Spectroscopy
Instrumentation

2. Processes of Deactivation
Internal
conversion
vibrational
relaxation
Intersystem
crossing
absorption
fluorescence
external
conversion
phosphorescence

3. Relationship between absorption
and fluorescence. The absorption
and fluorescence emission spectra
are represented by the solid and
dashed lines, respectively. The
fluorescence transitions occur
generally at longer wavelengths
because the energy differences
are less.

4. Excitation
Wavelength
selector
Sample
cell
Excitation
source
Emission
Wavelength
selector
Detector
Signal processor
Block diagram of a molecular
photoluminescence spectrometer.
Readout

5. If only filters are used for wavelength selection,
the instrument is designated a fluorometer.
In a spectrofluorometer, monochromators are
employed.
Typical optical configurations for both types
of instruments

6. Fig.1 (a)

7. Fig.1 Optical diagrams of a typical fluorometer
and a typical single-beam spectrofluorometer.
Fluorometer. Apertures are used to define
the width of the excitation beam entering the
sample cell and the width of the emission beam
viewed by the detector.

8. Fig.1(b)

9. Fig.1(b) the spectrofluorometer.
A mirror is used to focus an image of the
source onto the entrance slit of the excitation
monochromator. One or more lenses, between
the exit slit of the excitation monochromator
and the cell, are used to image a narrow beam
of excitation radiation into the cell. Another lens
or combination of lenses collects and images
the sample fluorescence onto the entrance slit
of the emission monochromator.

10. Excitation source
Under most conditions, the photoluminescence
signal is directly proportional to the incident
radiant power.
The ideal excitation source
a stable and high radiance at the excitation
wavelengths of interest

11. For simple fluorometers
the most common source:
a low-pressure mercury arc lamp
The intense lines at 254, 312, and 365 nm
are suitable excitation wavelengths for many
Molecules.

12. For commercial spectrofluorometers:
the most common source:
75- to 450 W high-pressure Xe arc lamps
high-pressure Hg and Xe-Hg lamps
Wavelength-Selection Devices
filter or monochromators
(to maximise the fluorescnece signal and
to minimise) the background signal)

13. Lasers It might be expected that the greater irradiance
of lasers compared to conventional sources would
be ideal for molecular phtoluminescence
measurement.
Detection limits with laser excitation are often
no better than those obtained with high-intensity
conventional sources if background luminescence
is dominant at the detection limit.

14. Lasers vs Conventional sources:
more expensive
more complex
more difficult to maintain
often less stable

15. Wavelength-Selection Devices
The central wavelength and bandpass of the
excitation and emission wavelength selectors
(filters or monochromators) are chosen.
to maximise the fluorescnece signal and
to minimise) the background signal)

16. Notes: to prevent the viewing of scattered source radiance,
the wavelength ranges passed by the excitation and
emission wavelength selectors should not overlap
increasing the excitation bandpass with continuum
sources has the benefit of increasing the incident
radiant power
because the emission band is usually broad ( nm),
increasing the emission bandpass results in a larger
fluorescence signal

17. Sample compartment and sample holder
Several different types of sample cells are used.
Test-tube-shaped cells are adequate for simple
fluorometers. More often, a standard square sample
cell is employed; they are polised on all sides, unlike
some absorption cells, which need only be polished
on two opposite sides.
Construction material:
high-quality synthetic silica ,quartz

18. Cell Geometry
lex
lem
Luminescence is viewed at 90oC with respect to the
excitation axis. Mirrors can be placed at the cell walls
opposite the excitation and viewing walls to increase
the detected luminescence signal by about 50%.

19. Detectors
photomultiplier tube

20. Fig (a)
Fig (b)

23. Compensation and correction techniques
Source stability
Wavelength dependence of the efficiency of
optical components
Affect
the magnitude of luminescence signals
calibration curve linearity
the magnitude and shape of luminescence
spectra

24. Source Intensity Compensation
drift or noise
Soln. Double-beam design

26. What is Self-quenching in Luminescence?
What is Self-absorption in Luminescence?