SATISH KUMAR.

J
PH.D STUDENT
MYSORE UNIVERSITY
Introduction
spectrometry
The principle of spectroscopy
Types of spectrometer
Data analysis
fluorescence
Principal of florescence
Advantages and disadvantages of flurometry
 In chemistry, spectrophotometry is the quantitative
measurement of the reflection or transmission
properties of a material as a function of wave length.
 It deals with visible light , near – ultra violet and near
infra red waves.
 The principle of spectrophotometry:
 The intensity of color is a measure of the amount of a material in
solution.
 A second principle of spectrophotometry is that every substance
absorbs or transmits certain wavelengths of radiant energy but
not other wavelengths.
 example-chlorophyll always absorbs red and violet light, while it
transmits yellow, green, and blue wavelengths. The transmitted
and reflected wavelengths appear green—the color our eye
“sees.”
 The light energy absorbed or transmitted must match exactly the
energy required to cause an electronic transition (a movement of
an electron from one quantum level to another) in the substance
under consideration. Only certain wavelength photons satisfy this
energy condition. Thus, the absorption or transmission of specific
wavelengths is characteristic for a substance, and a spectral
analysis serves as a “fingerprint” of the compound.
 LIGHT AND THE PERCEPTION OF COLOR
 Light is a form of electromagnetic radiation. When it
falls on a substance, three things can happen:
 The light can be reflected by the substance • it can be
absorbed by the substance
 Certain wavelengths can be absorbed the remainder
transmitted or reflected.
 The hearth of the spectrophotometer is the absorbance
and transmittance of light.
 The Beer-Lambert law (or Beer's law) is the linear
relationship between absorbance and concentration of
an absorbing species. The general Beer-Lambert law is
usually written as:
 A = a() * b * c
 where A is the measured absorbance, a() is a
wavelength-dependent absorptivity coefficient, b is the
path length, and c is the analyte concentration.
Development of FT-IR Spectroscopy for Microbial Identification
 Fourier transform–infrared (FT-IR) spectroscopy has been used in
industrial applications such as chemical and pharmaceutical manufacturing
and quality control for decades to provide positive fingerprint identification
of materials, but only more recently has been applied to the challenge of
microbial identifications.
 FT-IR microbial identification is based on the fact that each molecule's
functional groups absorb infrared radiation to generate a characteristic
absorption or transmission spectrum that is rich in information and unique
to that molecule.
 Spectra can be analyzed or searched against libraries of reference materials
to positively identify unknown materials, Just like human fingerprints
uniquely identify their owner,
 IR spectroscopy provides a spectral fingerprint that uniquely identifies a
chemical compound. In the field of microbiology, an FT-IR spectrum
reveals a cell's fingerprint, which reflects its biochemical composition
including proteins, lipids, DNA and RNA, and carbohydrates. The
method's high sensitivity and selectivity make it possible to identify
microorganisms even down to the strain level.
 Data Analysis
The evaluation of the microorganism spectra should be undertaken using the first derivative
of the resulting spectra. Broad overlapping bands characteristic of microorganism spectra
become better resolved by taking the first derivative. Taking the first derivative of the spectra
also provides a simple baseline correction. The intensity of the bands in the first derivative
spectra is approximately 100 times smaller than in the original spectra. Measured spectra can
be evaluated using either cluster analysis or an identity test report.

Cluster analysis helps identify similarities between the spectra of microorganisms under
analysis and the spectra of known species and can be most useful for differentiating
spectroscopically similar strains. The wave number regions, weighting factors, and
mathematical methods used for cluster analysis must be specified. Two very popular cluster
analysis algorithms for microbial identification are average linkage and Ward's algorithm. A
dendrogram is a tree diagram frequently used to illustrate the arrangement of the clusters
produced by a clustering algorithm. In the average linkage method, the fusion values in the
dendrogram correspond at least approximately to the original distance values in the spectra,
making the dendrograms relatively easy to interpret.


 Comparison with Spectral Libraries :
 Established and reliable reference techniques have been
used to develop spectral libraries of microorganisms.
These libraries are important for the identification and
classification of microorganisms. Repeat measurements
are carried out over time of independently prepared
strains of the sample cultivated under identical
conditions in different batches of the same medium. A
valid spectral database should cover a high number of
different species with a high number of reference
strains for each species. The composition should be
well balanced, and all reference strains should be
identified carefully by independent methods.
Some of the advantages and disadvantages of using FT-IR
spectroscopy methods for analyzing microorganisms are as
Advantages
 1.Relatively fast and simple to use: Little or no sample preparation required for
spectral acquisition.
 2.Sensitive method that requires very little sample: ng-μg.
 3.Nondestructive: The bacterial cell remains intact during analysis.
 4.Universal method: The instrument and software are readily available and can
be used for routine analysis.
 5.Qualitative as well as quantitative analysis: Spectra provide information
about bacterial cell composition and
 quantify the number of bacteria or amount of functional groups present in a
sample.
 6.Multiple sample environment: Samples in the form of liquid, gas, powder,
solid, or film can be tested.
 7.Identification and discrimination of bacteria: Bacteria can be discriminated
based on their physiological state such
 as live, dead, injured, and treated.
 8.Relatively less expensive for bacterial identification compared to several
commonly used methods.
 Disadvantages
 1.Environmental conditions around the FT-IR instrument
can cause variations in the spectra, hence background
 scans and multiple scans of the same sample are required.
 2.Complex samples like mixtures of bacteria produce
overlapping spectra, which may lead to misinterpretation of
 results. Hence a bacterial separation or purification step is
required in some instances.
 3.A complete library of spectra for each type of bacteria is
recommended to facilitate detection.
 4.May require standardization, rigorous data collection, and
expertise in the chemometric analysis of spectra.
 5.Culture medium, growth time, and growth temperature
may cause variations in spectra.
 6.Presence of water in a sample may influence the bands at
certain specific wavenumber.
 Conclusion
spectroscopy is a whole-cell fingerprinting technique
that provides high sensitivity, subspecies specificity,
and applicability to all microorganisms. Further, it can
be used to identify and classify microorganisms by
genus, species, and strain utilizing the well-established
mathematical methods of cluster analysis and
quantitative searching of reference data sets.
 Fluorescence spectroscopy (also known as
fluorometry or spectrofluorometry) is a type
of electromagnetic spectroscopy that
analyzes fluorescence from a sample. It
involves using a beam of light,
usually ultraviolet light, that excites the
electrons in molecules of certain compounds
and causes them to emit light; typically, but
not necessarily, visible light.
 Molecules have various states referred to as energy levels. Fluorescence
spectroscopy is primarily concerned with electronic and vibrational states.
Generally, the species being examined has a ground electronic state (a low energy
state) of interest, and an excited electronic state of higher energy. Within each of
these electronic states there are various vibrational states.
 In fluorescence, the species is first excited, by absorbing a photon, from its
ground electronic state to one of the various vibrational states in the excited
electronic state. Collisions with other molecules cause the excited molecule to lose
vibrational energy until it reaches the lowest vibrational state of the excited
electronic state.
 The molecule then drops down to one of the various vibrational levels of the
ground electronic state again, emitting a photon in the process. As molecules may
drop down into any of several vibrational levels in the ground state, the emitted
photons will have different energies, and thus frequencies. Therefore, by analysing
the different frequencies of light emitted in fluorescent spectroscopy, along with
their relative intensities, the structure of the different vibrational levels can be
determined.
 In a typical fluorescence (emission) measurement, the excitation wavelength is
fixed and the detection wavelength varies, while in a fluorescence excitation
measurement the detection wavelength is fixed and the excitation wavelength is
varied across a region of interest. An emission map is measured by recording the
emission spectra resulting from a range of excitation wavelengths and combining
them all together. This is a three dimensional surface data set: emission intensity
as a function of excitation and emission wavelengths, and is typically depicted as
a contour map.
 Two general types of instruments exist: filter
fluorometers that use filters to isolate the incident light
and fluorescent light and spectrofluorometers that use
a diffraction grating monochromators to isolate the incident
light and fluorescent light.
 Both types use the following scheme:
The light from an excitation source passes through a filter
or monochromator, and strikes the sample. A proportion of
the incident light is absorbed by the sample, and some of
the molecules in the sample fluoresce. The fluorescent light
is emitted in all directions. Some of this fluorescent light
passes through a second filter or monochromator and
reaches a detector, which is usually placed at 90° to the
incident light beam to minimize the risk of transmitted or
reflected incident light reaching the detector.
 Various light sources may be used as excitation sources, including lasers, LED, and
lamps, xenon arcs and mercury-vapor lamps in particular. A laser only emits light of
high irradiance at a very narrow wavelength interval, typically under 0.01 nm, which
makes an excitation monochromator or filter unnecessary.
 Filters and/or monochromators may be used in fluorimeters. A monochromator transmits
light of an adjustable wavelength with an adjustable tolerance. The most common type
of monochromator utilizes a diffraction grating, that is, collimated light illuminates a
grating and exits with a different angle depending on the wavelength. The
monochromator can then be adjusted to select which wavelengths to transmit. For
allowing anisotropy measurements the addition of two polarization filters are necessary:
One after the excitation monochromator or filter, and one before the emission
monochromator or filter.
 The detector can either be single-channeled or multichanneled. The single-channeled
detector can only detect the intensity of one wavelength at a time, while the
multichanneled detects the intensity of all wavelengths simultaneously, making the
emission monochromator or filter unnecessary. The different types of detectors have
both advantages and disadvantages.
 The most versatile fluorimeters with dual monochromators and a continuous excitation
light source can record both an excitation spectrum and a fluorescence spectrum. When
measuring fluorescence spectra, the wavelength of the excitation light is kept constant,
preferably at a wavelength of high absorption, and the emission monochromator scans
the spectrum. For measuring excitation spectra, the wavelength passing though the
emission filter or monochromator is kept constant and the excitation monochromator is
scanning. The excitation spectrum generally is identical to the absorption spectrum as
the fluorescence intensity is proportional to the absorption.[5]
 Advantages of fluorometer: - Very specific -Very
sensitive. - Wide Concentration Range - Simplicity and
Speed-LowCost.

 Disadvantages: The fluorescence is very sensitive to

Environmental changes which include: PH,
temperature, solvent contamination and UV light used
for excitation can photochemical change
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