Contents
Refractometry
Principle
Instrumentation
Applications
Fluorescence and Phosphorescence
Principle
Instrumentation
Applications
Refractometry
Refraction: Change of light path, when it pass from one medium to another
Angle of
incidence
Snell’s Law Refractometry is an analytical
method for measuring
refractive index
n= refractive index=
Angle of
refraction c is velocity of light in vacuum
v is velocity of light in medium
[Link]
Critical Angle and Total Internal Reflection
[Link]
Abbe Refractometer
It is based on critical angle
Sample is contained as a thin layer
(~0.1mm) between two prisms
The illuminating prism face is rough-
ground: when light is reflected into the
prism, this surface effectively becomes
the source for an infinite number of rays
that pass through the sample at all angels
The eyepiece of the telescope is
provided with crosshairs: in making a
measurement, the prism angle is
changed until the light-dark interface
just coincides with the crosshairs
The position of the prism is then established from
the fixed scale (which is normally graduates in
units of nD) [Link]
[Link]
Two Amici prisms that can be rotated
with respect to another serve to collect
the divergent critical angle rays of
different colors into a single white
beam, that corresponds in path to that
of the sodium D ray
[Link]
Applications of Refractometry
1. Identity of sample by comparing its refractive index to known values
2. Purity of the sample by comparing its refractive index to the know value
3. Concentration of the solution by comparing refractive index to standard
curve
[Link]
Luminescence
Luminescence is an emission of ultraviolet, visible or infrared photons
from an electronically excited species
Possible de-excitation pathways
IC Internal Conversion
ISC Intersystem Crossing
S Single state
Jablonski diagram T Triplet state
Stokes Shift and Quantum Yield
Stokes shift is the gap between the maximum of the first absorption band and the
maximum of the fluorescence spectrum (expressed in wavenumbers)
Instrumentation-Fluorescence
Fluorimeter schematic
Usually, fluorescence is collected at an
angle of 90 degrees with respect to the
optical axis set by the excitation light
beam. This geometry maximizes the
efficiency of the emission collection and
reduces the background due to the
excitation light.
Generally, Cuvettes are used as sample cell [Link]
Instrumentation-Phosphorescence
Phosphorescence is not usually observed
in fluid solutions near room temperature.
One reason for the absence of
phosphorescence is the long
phosphorescence lifetimes and the
presence of dissolved oxygen and other
quenchers
In practice, dissolved quenchers and
nonradiative decay rates result in
vanishingly small phosphorescence
quantum yields in room-temperature
solutions
Hence, phosphorescence is measured at
low temperature. For example, liquid
nitrogen is used for this purpose.
Applications of Fluorescence Spectroscopy
Bioscience
High precision quantification of DNA and RNA, dynamics of biomolecules, protein-
protein interactions, protein-DNA interactions, enzymatic reaction mechanisms etc…..
Environmental
Used in detection of air-borne and water borne pollutants and analysis of impurities in
petroleum products.
Pharmaceutical
Spectrofluorometric techniques are also used in the pharmaceutical field to analyze drugs.
An example is the analysis of co-formulated tablets prescribed as cholesterol medication.
Synchronous fluorescence spectroscopy provides a simple, fast, and accurate method for
analyzing a tablet called Atoreza©, which contains both Ezetimibe and Atorvastatin
calcium.
Forensic Applications used in detection of pigments and dyes found in a paint sample,
detection of LSD, analysis of ink, fibres and tape materials. Most importantly used in
visualization of latent fingerprints with the help of laser and fluorescent powder.
[Link]
Applications of Phosphorescence Spectroscopy
Room Temperature Phosphorescence Spectroscopy has various applications
Photodynamic Therapy Based on Room-Temperature Phosphorescence Probes
Speleothem Room-Temperature Phosphorescence Dating(alternative approach to
radiocarbon dating when no organic matter (seed, wood, bone, etc.) is available or
when the expected age of the sample is greater than the limits of radiocarbon
dating)
Pressure-Sensitive Paints in Automotive Model Testing and Aerodynamics
Applications
Monitoring of Oxygen Profiles in Packaged Foods
Latent Fingerprint Visualization
Garcia [Link], Applied Spectroscopy Reviews, 42: 605–624, 2007