Advance Pharmaceutical Analytical

Techniques

SEMINAR BY,
S.REKHA
•Two primary kinds of instruments to measure
the fluorescence
Filter Fluorimeter,
Spectrofluorometer.
 INSTRUMENTATION:
SOURCE OF LIGHT

FILTERS AND
MONOCHROMATORS

SAMPLE CELLS

DETECTORS
Fluoromter components
1. SOURCE OF LIGHT:

Mercury vapour lamps

Xenon arc lamps
Tungsten lamps
Lasers
LED’s
1.Source of light:
i. Mercury vapour
lamp:
• The intensity is
concentrated in
wavelength of the Hg
spectrum.
• Low fill gas pressure(10
torr)
ii. Xenon arc lamp
Providing light out put
from 190-1200 nm
10-30 atmosphere.
Versatile and powerful.
iii. Laser Sources:

•Laser --- Light

Amplification by Stimulated
Emission of Radiation.

• Mechanism – inverted
population of energy
states.
The wide tuning ranges of
external cavity diode lasers
provide a variety of
wavelength and their narrow
line width continuous
tunablity leads to high
resolution scanning
capability.
iv. LED’s:
LED’s based on
A1InGap and InGaN.
Spectra for blue,
yellow-green, and red
LEDs.
The spectral bandwidth
is approximately 25nm
for all three colors.
2. Filter and monochromators:
In filter Fluorimeter,
Primary filter - absorbs visible radiation and transmits
uv radiation.
Secondary filter - absorbs uv radiation and transmits
visible radiation.

• Filters are of two types:

1.Absorption filter,
2.Interference filter.
Absorption filters:
Absorption filters ---
normally made of color
glass,
Typically used includes
long pass and short pass
cut-off filters.
Interference filters:
•Monochromator:

1.Entrance slit – to get a narrow slit.

2.Collimator – produces a parallel beam of radiation.
3.Diffraction grating – to disperse radiation.
4.Focuser – reforms the image of the entrance slit and focuses
it on a planar surface called focal plane.
5.Exit slit –to fall on the sample detector.
In Spectrofluorometers,

Excitation monochromator - provides a suitable radiation

for excitation of molecule.
Emission monochromator – isolate only the radiation
emitted by the florescent molecule.

Gratings are of two types:

 1. Diffraction gratings,
 2. Transmission gratings.
Diffraction gratings:
Diffraction produces reinforcement.
Transmission gratings: Refraction produces
reinforcement.
λ = d sinθ
m

Where λ = wavelength of radiation produced.

d =1/lines per cm.
m =order of(0,1,2,…)
θ =angle of incidence or diffraction.
3. Sample cell/cuvette:
Borosilicate or quartz
glass or various plastics.
10 mm square cuvettes
and / or 13 or 25 mm test
tube.

Adaptors are available for

9 ml capillary tubes and
100 ml mini cells for
small volumes.
Falling stream flow cell:
Fluorometers are also
available for flow-
through studies.
where samples are
pumped through a flow
cell in the instrument’s
sample chamber.
This allows for
continuous, on-line
monitoring of samples.
4. Detectors:
 When a radiation is passed through a sample cell, part of it
get absorbed by the sample solution and the rest is
transmitted .
 This transmitted radiation falls on the detector and the
intensity of absorbed radiation can be determined.
 Light energy → electrical signal → recorded.
 Detectors used in flourimetry is
Photomultiplier tubes
Photomultiplier Tube (PMT)
PMT
 Used to provide several
orders of gain (106)
Includes “several”
intermediate anodes (dynodes)
• Each is given a voltage
higher than the previous
one
• e- arrives with enough
energy to eject multiple
electrons
Photomultiplier Tube (PMT):
Advantages
Standard device
Large signals
Large active area possible
Fast rise time possible
Disadvantages
Large physical dimension
High voltage required
Gain instability as a function of temperature
Sensitive to magnetic fields
Instruments:
1. Fluoromter:
 a. single beam (filter) flourimeter.
 B. double beam (filter) flourimeter.
2. Spectrofluorimeters:
 a. those containing of flourscence attachment for a
spectrophotometer.
 b. self contained instruments usually with two
monochromators.
Single beam (filter) Fluorimeter
Single beam (filter) Fluorimeter:

Source: mercury lamp.

Optical system composed of primary filter.
The emitted radiation (fluorescent radiation) is
measured at 90°, by using a secondary filter.
Advantages:
Simple in construction,
Cheaper and easy to operate,
The range of application can be widened by using different
combinations of primary and secondary filters.

Disadvantages:
It is not possible to use sample and reference solution at a time.
Rapid scanning to get excitation or emission spectrum of the
compound is not possible.
Double beam filter flourimetry
Double beam filter flourimetry:
The two incident beams from a single light source pass through
primary filters separately and fall on the either sample or
reference solution.
The emitted radiation from sample or reference passes
separately through second filter and produces combined
response on a detector.
Advantages:
Sample and standard solution can be analysed simultaneously.

Disadvantages:
Expensive one.
Rapid scanning is not possible.
Spectrofluorometers:
Double beam spectroflourimetry:
The primary filter in double beam (filter) flourimetry is replaced by
excitation monochromator
The secondary is replaced by emission monochromator.
The detector is photo multiplier tube.
The fluorescent intensity was recorded by detector using
Spectrofluorometer we can know,
 the wavelength of best excitation
 the wavelength of strongest emission.
Advantages:
Rapid scanning,
More sensitivity,
more accuracy,
continuous reading,
latest and precise manner results.
Quenching of fluorescence
Quenching of fluorescence and types:

Introduction:

Any process which decrease the fluorescence

intensity of the sample
Excited state reactions
Molecular rearrangements
Energy transfer
Ground-state complex formation
Collisional Quenching
Quenchers:

Oxygen – undergoes intersystem crossing

Aromatic and aliphatic amines – charge transfer reactions
Iodine and Bromine – intersystem crossing, spin-orbit
coupling of excited state fluorophore and halogen
Electron scavengers - protons, histidine, cysteine, NO-,
fumarate, Cu2+ , Pb2+ , Cd2+ , Mn2+
Acryl amide
Purines and Pyrimidines – FAD and NADH quenched by
adenine group
Selective quenching of a given fluorophore
§Types of quenching:

Concentration quenching.
Chemical quenching,
Collisional quenching,
Static quenching.
1.Self quenching or concentration quenching:

Low concentrations (μg or ng) - linearity is observed.

High concentrations (mg/ml) of the same substance
proportionate increase in fluorescence intensity does not
occur.
2. Chemical quenching:

 PH
 Halides
 Electron withdrawing group
 Heavy metals
pH: aniline at pH = 5-13 blue fluorescence 290 nm

aniline at pH<5
aniline at pH>5
No fluorescence
Oxygen:
Presence of oxygen paramagnetic property

triplet ground state

quenching
3.Static quenching:
When the quencher (Q) forms a stable complex with the
fluorophore in the ground state and this complex is
inherently non-fluorescent.
The remaining uncomplexed fluorophore emit normally
with the same quantum yield and lifetime as in the
absence of the quencher.

(e.g.) caffeine reduces the fluorescent intensity of

riboflavin, by complex formation.
4. Collisional quenching:
It is the result of several factors like presence of
halides, heavy metals, increased temperature and
decrease in viscosity, where numbers of collisions
are increased. Hence quenching take place.

M* → M + hν1 Fluorescence
M* + Q → M + Q + heat Quenching
Thank you