4.copy of Spectroscopy - Final

SPECTROSCOPY
• The study of interaction of electromagnetic radiation with, matter.

• Quantitative and qualitative analysis.
• Spectrometer An instrument designed to measure the amount and wavelength distribution of
light either absorbed or emitted by a sample
• Spectroscopy A set of techniques which measure the amount and wavelength distribution of light
either absorbed or emitted by a sample.
Electromagnetic radiation
• According to quantum mechanics,
EMR has particle and wave
nature.
• Interaction of electromagnetic
radiation with matter- dependent
upon both the properties of the
radiation and the appropriate
structural parts of the samples
involved.
• EMR- typically described by
physical properties wavelength,
frequency and photon energy
• Wavelength (nm) λ is the spatial distance
between two consecutive peaks (one
cycle). • The energy E, of a photon can be
• Frequency (1 Hz) ν is the number of related to its wavelength and
oscillations made by the wave within the frequency with the help of
timeframe of 1 s. Planck's constant, h.
• Radiation of all wavelengths travel at a
single speed.
• Speed of light, denoted c, is dependent
on the substance through which it travels,
and reaches a maximum in a vacuum. • h = 6.63 x 10-34 Js
• The speed of light is a constant of • c = 3 x 108 m s-1
proportionality between frequency and
wavelength
c = νλ
Beer-Lambert law
The amount of light absorbed (absorbance or extinction) is proportional to the
concentration of the absorbing substance and to the thickness of the absorbing
material (path-length).
Io/l is known as the absorbance or the optical density (O.D.).
I/Io is known as the transmittance, T
a is the absorptivity or the absorption coefficient.

b = the absorbing thickness/path length
C = the concentration of the absorbing material.
→ optical density is proportional to the sample concentration if the path-
length is constant.
• Preparation of a calibration curve from a number of standards.

• Plotting known concentrations of a given substance against absorbance
at a particular wavelength (usually the λmax).
UV-Visible Spectroscopy
• Works on the phenomenon of electronic transitions in molecules in the
visible and UV regions of the electromagnetic spectrum.
• Used quantitatively to determine the concentration of an absorbing
species in solution using the Beer-Lambert law
• UV and visible light are energetic enough to shift outer electrons to
higher energy levels.
• Molecular (sub-)structures responsible for interaction with
electromagnetic radiation are called chromophores.
Absorption Spectrum
• The pattern of energy absorption by a
substance when light of varying
wavelength passes through it is
uniquely characteristic of the
substance.
• Transmittancy or optical density of a
A/c to quantum theory in order to be
particular concentration of the
excited the electron will accept only
substance is measured at different
that radiation which has the
wavelengths keeping path length
exact quantized energy to push it
constant
into a permitted energy level.
e.g., hydrogen electron- 1.217 Ao
Chromophores in proteins
• peptide bond- intense peak at 190 nm
• Tyrosine and tryptophan dominate the
typical protein spectrum with their
absorption maxima at 274nm and 280 nm,
respectively.
• Phenylalanine weak absorption maximum at
257 nm
• Prosthetic groups (e.g. haem, flavin,
carotenoid)- contain three maxima in the
visible region of the electromagnetic
spectrum (~ 420 nm, 450 nm, 480 nm).
Chromophores in genetic material
• All nucleotides absorb UV light and
are characterized by a strong
absorption at wavelengths near
260 nm.
• Electronic transitions of the purine
and pyrimidine bases that occur
between 260nm and 275 nm.
Instrumentation
2 types- Single beam and dual-beam spectrometers
• Source
• Filters/Monochromator
• Sample compartment
• Detector
• Recorder
Applications
Qualitative and quantitative analysis
• To identify certain classes of compounds both in the pure state and in biological
mixtures (e.g. protein-bound).- by plotting absorption spectrum curves.
• Most commonly used for quantification of biological samples either directly or via
colorimetric assays.
Enzyme Assay
• Quantitative assay of enzyme activity is possible when the substrate or the product is
colored or absorbs light in the ultraviolet range.
• the rate of appearance or disappearance of a light absorbing product or substrate can
be followed with a spectrophotometer.
• Continuous record of the progress of the reaction
Molecular weight determination
where w is the weight of the compound in grams per litre. and b is the pathlength.
• Molecular weights of only small molecules may be determined by this method.
Study of Cis-Trans Isomerism
• The absorption spectra of the isomers are different
Control of Purification
• Impurities in a compound can be detected by experimentally verifying whether the
given compound shows an absorption maxima not characteristic of it.
Difference spectroscopy
• Sensitive method for detecting small changes in the environment of a chromophore.
• Difference spectroscopy involves comparison of absorption spectra of two samples which
differ only slightly in their physical states.
Turbidimetry and Nephelometry
• Turbidimetry - routinely used to measure the number of bacteria in a given suspension.
• Measurement of the intensity of this transmitted light will allow one to have an idea of the
number of particles in the suspension.
• Nephelometry- measure the intensity of
the light scattered by the particles in
suspension.
REFERENCES
1. Principles and Techniques of Biochemistry and Molecular Biology: Keith Wilson and John
Walker
2. Biophysical Chemistry: Principles and Techniques: Upadhyay, Upadhyay and Nath.
3. Lewis, S.W. (2013). Encyclopedia of Forensic Sciences || Spectroscopy: Basic Principles. ,
635–640. doi:10.1016/B978-0-12-382165-2.00256-7

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SPECTROSCOPY

• The study of interaction of electromagnetic radiation with, matter.

a is the absorptivity or the absorption coefficient.

• Preparation of a calibration curve from a number of standards.

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