SPECTROFLUORIMETRY INSTRUMENTATION

Trainer: Chandramouli R

A generalized Spectrophotometry consists of:

Source of radiation A sample cell Photodetector Read out device

A generalized Fluorimeter/ Spectrofluorimeter consists of:

Source of radiation A primary filter / excitation Monochromator A sample cell A secondary filter / emission Monochromator Photodetector Read out device

FILTER FLUORIMETER
Primary filter Secondary filter

Absorbs Visible radiation and transmits UV radiation

Absorbs UV radiation and transmits Visible radiation

SPECTRO FLUORIMETER
Primary / Excitation Monochromator Secondary / Emission Monochromator

Provides a suitable radiation for excitation of molecule

Isolates only the radiation emitted by the fluorescent molecule

BLOCK DIAGRAM
Source of light Primary filter / Monochromator Secondary filter / Monochromator

SAMPLE

Emitted radiation from source

Resultant Specific DETECTOR luminescence wavelength of radiation Radiation emitted by the fluorescent molecule

Advantage of Spectro Flourimeter over Filter Flourimeter

Spectroflourimeter can measure the spectral distribution of Flourescence emission (emission spectrum) And the variation in emission spectral radiance with excitation wavelength (excitation spectrum)

INSTRUMENTATION

Source of radiation A primary filter / excitation monochromator A sample cell A secondary filter / emission monochromator Detectors Read out device

INSTRUMENTATION

Source of radiation A primary filter / excitation monochromator A sample cell A secondary filter / emission monochromator Detectors Read out device

Source of light
Source of light should emit a radiation over a continuous region and be of adequate intensity and stability Mercury vapour lamp Xenon arc lamp Tungsten lamp

INSTRUMENTATION

Source of radiation A primary filter / excitation monochromator A sample cell A secondary filter / emission monochromator Photodetector Read out device

Filters and Monochromators

Primary filter/Monochromator selects specific bands or wavelengths of radiation from the source and directs them through the sample in the sample cell Secondary filter/Monochromator Resultant luminescence from primary filter/monochromator is isolated by the secondary filter or emission monochromator and directed to the photodetector which measures the power of the emitted radiation.

INSTRUMENTATION

Source of radiation A primary filter / excitation monochromator A sample cell A secondary filter / emission monochromator Detectors Read out device

Sample cells

Cylindrical /Quadrangular in shape Path length 10mm or 1cm

Sample cells (contd) Types :

1. Right angle method (90) 2. Frontal method (37) 3. Rotating cell method
Monochromatic excitation light 90 sample

Monochromatic excitation light

37

sample

Instrumentation

Source of radiation A primary filter / excitation monochromator A sample cell A secondary filter / emission monochromator Detectors Read out device

Detectors

Photovoltaic cell Phototubes Photomultiplier tubes BEST & ACCURATE

PHOTOVOLTAIC CELL / BARRIER LAYER CELL

A Steel support (positive contact) B SeleniumSemiconductor layer C Metallic Layer coated with silver or gold D Negative contact E Metallic spray coating to protect from corrosion

PHOTO TUBES / PHOTO EMISSIVE CELLS

PHOTO MULTIPLIER TUBE

PHOTO MULTIPLIER TUBE