flourimetry ppt
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Transcript of flourimetry ppt
Advance Pharmaceutical Analytical Techniques
SEMINAR BY, S.REKHA
Two primary kinds of instruments to measure the fluorescenceFilter Fluorimeter, Spectrofluorometer.
INSTRUMENTATION:SOURCE OF LIGHT FILTERS AND
MONOCHROMATORSSAMPLE CELLS DETECTORS
Fluoromter components
1. SOURCE OF LIGHT:Mercury vapour lamps Xenon arc lamps Tungsten lamps Lasers LEDs
1.Source of light: i. Mercury vapourlamp: The intensity is
concentrated in wavelength of the Hg spectrum. Low fill gas pressure(10 torr)
ii. Xenon arc lampProviding 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. LEDs:LEDs 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 radiationemitted by the florescent molecule.
Gratings are of two types:
1. Diffraction gratings, 2. Transmission gratings.
Diffraction gratings:Diffraction produces reinforcement.
Transmission gratings: Refraction producesreinforcement. = dm
sin
Where = wavelength of radiation produced. d =1/lines per cm. m =order of(0,1,2,) =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 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)
PMTUsed 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):
AdvantagesStandard device Large signals Large active area possible Fast rise time possible
DisadvantagesLarge 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 sampleExcited 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 (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 pH5
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
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