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

LEDs

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.

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:
Entrance slit to get a narrow slit. Collimator produces a parallel beam of radiation. Diffraction grating to disperse radiation. 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. 1. 2. 3. 4.

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 instruments 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)

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 (g 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 + h1 Fluorescence M* + Q M + Q + heat Quenching

Thank you