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
m

sinθ

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 flowthrough 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 Energy transfer

Molecular rearrangements 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